Oils From Microorganisms

5
Oils from Microorganisms

James P. Wynn1 and Colin Ratledge2
1
Martek Biosciences Corporation

Columbia, Maryland
2
Lipid Research Centre, University of Hull,
Hull, United Kingdom
1. GENERAL INTRODUCTION AND BACKGROUND

INFORMATION
As this chapter is the only one in this new edition that is solely devoted to the potential use of microorganisms as sources of oils and fats, we have felt it appropriate to
preface our review by including a brief outline of the importance of microbial biotechnology in general so that the extent to which microorganisms can now be grown
and the amounts of products that are produced can be appreciated by the general
reader who may not be familiar with the field of microbial biotechnology.
1.1. Microorganisms and Biotechnology
Microorganisms range from bacteria, which are known as the prokaryotic microbes
as they do not contain a defined nucleus, although they do, of course, contain DNA,
to the eukaryotic microorganisms that do have a nucleus and range from the simple
yeasts to fungi, which may show more complex structural variations and be capable
of some limited differentiation. Some fungi can differentiate into macromolecular
forms and give rise to, for example, the mushrooms and toadstools. There are also a
range of microorganisms in the aquatic environments, and these will include both
Baileys Industrial Oil and Fat Products, Sixth Edition, Six Volume Set.
Edited by Fereidoon Shahidi. Copyright # 2005 John Wiley & Sons, Inc.
121
122
OILS FROM MICROORGANISMS
prokaryotic and eukaryotic forms. Many of these will only grow in the presence of
light (phototrophs), although a few will grow in the dark if some form of fixed carbon (e.g., sugar) is supplied (i.e., they will grow heterotrophically like other microorgansisms). Thus, the term microbe applies to a very wide range of living cells,
although all would be regarded, at least in the first instance, as being unicellular
and, as such, could only be seen as individual cells when examined down a microscope.
The advantages of microorganisms, which are used extensively in biotechnology
systems, are as follows:
The range of products that they can producefrom simple molecules like
ethanol and citric acid, to complex proteins, carbohydrates, etc.
They can be grown phenomenally fast: some bacteria can divide once every
10 minutes, although yeasts and fungi may take several hours to accomplish
this doubling of cell numbers;
They can be randomly mutated, by chemicals and other mutagens, so that
products can be produced in vastly increased quantities. Penicillin, for
example, when first produced, was at a few milligrams per liter; now the
current mutated strains will produce up to 100-g penicillin/L.
Microorganisms can also be modified by genetic manipulation where genes
(that is, specific sections of DNA coding for the synthesis of particular
proteins) taken from other living cells, i.e., other microorganisms, plants, or
even animals cells, can be easily incorporated into a microorganism so that it
will now produce not only its own proteins, but also the foreign protein.
Microorganisms can therefore produce, at least in theory, any product that we
currently can identify in any living cell. They will produce them faster, more safely
(you do not need to spray them with pesticides, herbicides, etc.) than any other
system. Moreover, the products can be produced to a guaranteed quality on a
year-round basis as productions do not depend on the vagaries of the weather or
climate. They are the ultimate controlled living system. This, then, is the world
of microbial biotechnology (1). The scale of cultivation can be in small fermenters
(about 15 m3) for production of very highvalue, lowvolume materials, such as
some of the current healthcare products, up to fermenters of 100500 m3, which can
be used to produce bulk materials such as citric acid, ethanol, and even whole cells
destined for animal or human foodthe so-called single cell proteins.
1.2. Microbial Oils

Microorganisms, like every other living cell system, produce lipids. All cells are
surrounded by membranes that require the synthesis of fatty acids (except in the
case of the most ancient of bacteriathe Archaeawhere long-chain, branched
terpenoid structures are used), which are then attached to glycerol 3-phosphate giving rise to the phospholipids and triacylglycerols. This system of fatty acid and lipid
GENERAL INTRODUCTION AND BACKGROUND INFORMATION
123
Figure 1. (a) Cells of an oleaginous yeast, Cryptococcus curvatus (formerly Candida curvata), in
which the lipid droplets constituting about 70% of the cell weight of the cells can be clearly seen.
(b) Electron micrograph of extracted oil from the same yeast shows a possible boundary layer
around the oil droplets. The oil is virtually pure triacylglycerol. (This figure is available in full color
at http://www.mrw.interscience.wiley.com/biofp.)
biosynthesis is then used, in some cases, for the overproduction of triacylglycerols

that then serve as reserve storage materials within the microbial cell (see Figure 1).
These are the so-called oleaginous species, and the oils are then not only useful to
the microorgansism to reuse during any subsequent period of starvation, but also
may be considered as sources of these commodities. The oils are referred to as
single cell oils (SCO), which is a euphemism similar to the term single cell protein used to indicate protein derived from microbial (single) cells. Single cell oils
is now a generally accepted term and is used in preference to microbial oil, which
may often create an undesirable impression with an otherwise unappreciative public.
Microorganisms have been considered as potential sources of oils since the early
decades of the twentieth century when scientists, particularly in Germany, began to
explore them as an alternative to plant oils, which were increasingly in short supply
because of the advent of two world wars. Indeed, during the Second World War,
124
processes had been developed that produced small amounts of the oils and fats,
although, because of the lack of large-scale fermentation technology, this never proceeded beyond a demonstration scale (2, 3). None of the microbial fat seems to
have been used for human consumption, however. Nevertheless, there was considerable interest in microbial oils and fats and in exploring their potential as alternatives to plant seed oils, which were poorly developed at this time.
Work on understanding which were the best oil-producing strains of microorganism to use and how they had to be cultivated to induce the highest possible lipid
contents was carried out during both war-time and peace-time in the first half of
the twentieth century, so that by the late 1950s, a shrewd understanding of the range
of oils that could be produced had been gained as well as knowing how these organisms had to be grown to produce the highest yields (2). However, the economic
value of the microbial oils was always the Achilles heel to their commercialization.
Advances in agriculture, particularly after the end of the Second World War in
1945, meant that all agricultural commodities, and not just oils and fats, became
cheaper and more plentiful than ever before. As the microorganisms being considered for oil production had to be grown on sugar, which was an agricultural product,
it was clear that it was nonsensical to grow one field of sugar cane (or sugar beet)
for it to be converted at the ratio of 5 tons of sugar to 1 ton of oil in an expensive
bioreactor (i.e., the fermenter) to produce oil that could be produced just as easily
using the self-same field to grow a plant oilseed crop.
Thus, by the 1960s, there seemed to be no arguable case in favor of microbial
oils being an economic proposition. And yet today, in the first decade of the twentyfirst century, we have at least four microbial oils that are, or have been, in full-scale
production, with the prospect that others may soon follow.
This change in attitude toward microbial oils has originated mainly, but not
exclusively, by the appreciation of the need for specific polyunsaturated fatty acids
(PUFAs) to be included in our diets, both for our infants and babies as well as ourselves in later life. Such polyunsaturated fatty acids, which have been known to
exist in microorganisms for many years, are now in demand. They cannot be easily
produced by plants and, in some cases, cannot be produced at all by plants. The
sources of these PUFAs then are from animals and, in particular, fish. Fish, though,
are a dwindling resource and the oils, which are mainly obtained from their livers
or the bodies of fatty fish, can be contaminated with various pollutants, including
organo-mercury compounds, dioxanes, and other materials that we would do best to
avoid in our own diets. Thus, the commercial exploitation of microbial oils has
originated by them being destined for human consumption and by them not being
readily available from traditional sources, either plants or animals.
Coupled with the realization that microorganisms can be a unique source of certain desirable oils, has been the advent of large-scale fermentation technology
throughout the latter half of the last century. This has culminated in the ability to
design, build, and operate purpose-designed fermenters in excess of 1,000,000-L
capacity for the production of microorganisms to provide a variety of products. A
simple calculation tells us that a 1000-m3 fermenter that produces 100-g cells/L in 4
days would yield a 100 tons of biomass with, perhaps, 40 tons of oil: an annual
Acid
Base Antifoam
125
CENTRIFUGE
Waste
cryovial
125 ml
To All Fermentors
SPRAY
DRYER
1L
20 L
200 L
2,000 L
12,000 L
120,000 L
PACKAGING
DRY BIOMASS
Figure 2. A diagrammatic presentation of a fermentation system for the production of a single

cell oil. The main fermenter is inoculated from a smaller vessel at about 10% (v/v). Additional
nutrients, including glucose, may be added to the microbial culture within the fermenter during
growth. The process operates in a batch mode so that when the cells reach their highest lipid
contents (see Figure 3), the fermenter is emptied, the cells are removed from the broth, and then
finally they are spray-dried. In this form, the oil within the cells is stable and can be extracted by
solvents whenever, and wherever, is convenient. (From 50, with kind permission of the author,
Dr. David J. Kyle, and the publishers.)
yield of over 3000 tons of oil. In agriculture, about 4 square miles (9 km2) of land
would need to be sown with sunflowers or oilseed rape to give the same yield of oil.
A typical large-scale process for the production of SCO is shown diagrammatically
in Figure 2. This would be typical of the current fermentation processes being used
for these types of products.
In this review, we aim to provide details of microbial oils that have been considered of commercial potential and to describe in more detail those microbial systems that are in commercial operation to provide key fatty acids for the expanding
nutraceuticals industry.
1.3. Microbial Lipid Production Systems
Microorganisms that accumulate more than 20% of their biomass are known as the
oleaginous species. This value, though, is arbitrary and has no precise numerical
definition. There are probably far more nonoleaginous species than oleaginous
ones; that is, most microorganisms will accumulate, even under the most propitious
conditions, only a few percent of their biomass as lipid. The lipid that is produced in
the oleaginous species is usually in the form of triacylglycerols (see Figure 1) and is
an intracellular reserve supply of both carbon and energy (and perhaps water) to
be used in times of nutrient starvation. The extent of lipid accumulation may range
from the lower limit of about 20% up to 70% of the cell weight being extractable
oil. The range of oil contents of a selected range of microorganisms is given in
Table 1 together with a profile of the constituent fatty acids. More detailed lists
TABLE 1. Lipid Contents and Fatty Acid Profiles of Selected Oleaginous Microorganisms (Compiled from Lists Given in (4)(6), Which
Should be Consulted, if Needed, for Further Information).
Major Fatty Acyl Residues
(Relative % w/w)
Organism
A. Yeasts
Candida sp. 107
Cryptococcus albidus
C. curvatus D1
Waltomyces lipofer2
Lipomyces starkeyi
Rhodosporidium toruloides
Rhodotorula glutinis
Trichosporon beigelii
Yarrowia lipolytica
B. Fungi
Zygomycetes
Conidiobolus nanodes
Notes :
Entomophthora coronata
Cunninghamella japonica
Mortierella isabellina
Rhizopus arrhizus
Mucor alpine-peyron
Ascomycetes
Aspergillus terreus
Fusarium oxysporum
Pellicularia practicolo
Lipid
Content
(% w/w)
14:0
42
65
58
64
63
66
Trace
Trace
Trace
Trace
Trace
Trace
44
12
32
37
34
18
5
1
4
6
3
72
45
36
Trace
Trace
Trace
37
12
11
26
43
60
86
57
38
57
34
39
16:0 16:1
(n-7)
18:0
18:1
(n-9)
18:2 18:3
(n-6) (n-3)
18:3
(n-6)
20:4
(n-6)
20:5
(n-3)
22:6
(n-3)
8
3
15
7
5
3
31
73
44
48
51
66
9
12
8
3
3
3
22
1
47
50
28
8
12
51
23
15
25
31
Trace
1
19
10
9
16
29
18
15
2
14
3
6
7
14
48
55
22
30
2
14
3
10
9
1
8
3
12
1
2
Trace
Trace
23
17
8
Trace 14
8
20
2
11
40
46
72
21
5
2
Others (%)
23:0 (3%)
24:0 (6%)
20:1 (13%)
22:1 (8%)
12:0 (40%)
20:0 (8%)
20:3 (6%)
Hyphomycetes
Cladosporium herbarum
Clavicipitaceae
Claviceps purpurea
49
Trace
31
12
35
18
60
Trace
23
19
12-HO18:1 (42%)
C. Microalgae and thraustochytrids3

Continued
Prokaryota
Spirulina maxima
Spirulina platensis
Eukaryota
Chlorella minutissima
Chlorella vulgaris
Crypthecodium cohnii 3
Isochrysis galbana
Monodus subterraneus
Nannochloropsis oculata
Phaeodactylum tricornutum
Porphyridium cruentum
Thraustochytrids
Schizochytrium sp.3
Thraustochytrium aureum3
22
25
8
1
63
26
4
23
9
10
12
21
13
16
16
10
19
15
10
30
2
5
21
2
1
11
10
22
21
5
28
52
50
23
20
45
24
5
12
16
12
1
58
21
3
5
3
1
<1
2
9
1
2
2
1
4
5
14
<1
1
1
<1
14
4
1
16
45
25
34
38
33
38
40
11
18:4 (11%)
14:1 (13%)
40
17
32
28
16
52
22.5 (n-5)
(8%)
25
1. This yeast was initially known as Candida curvata, then was renamed Apiotrichum curvatum, and now is regarded as Cryptococcus curvatus.
2. Formerly known as Lipomyces lipofer.
3. Microalgae cultivated phototrophically except for C. cohnii, Schizochytridium sp., and T. aureum, which are nonphotosynthetic and are grown heterotrophically.
128
are provided in other reviews (46) and in an earlier comprehensive monograph on

microbial lipids (7).
As SCO are usually triacylglycerols that can account for over 90% of the total
lipid in the microbial cell, this makes them potential substitutes for plant oils and
animal fats, although clearly their economic potential will rest on their intrinsic
value. It is to be emphasized, however, that an SCO will only complete with other
commercial sources if it can be shown to be better in some respect or cheaper than
the traditional source.
The biochemical procedure by which microorganisms accumulate lipid is now
understood in some detail and has been recently described (8). Readers wishing
more detailed information should consult this article. In brief, oleaginicity, which
is not shared by all microorganisms, depends on the presence of just a few key
enzymes.
The process of lipid accumulation begins by the cells being grown in a culture
medium in which the supply of a nutrient; usually it is nitrogen in the form of an
ammonium salt, becoming exhausted (this is shown schematically in Figure 3). At
the same time, there remains in the medium a surfeit of carbon, usually glucose or
some other assimilatable carbon source. The cells, as a consequence of this nutrient
exhaustion, can no longer grow and multiply, but they do continue to take up the
sugar present in the medium. It is this surplus sugar that then becomes the source of
carbon for lipid biosynthesis.
Figure 3. Stylistic presentation of the course of lipid accumulation by an oleaginous

microorganism. The concentration of nitrogen (NH3) in the medium is adjusted so that it
becomes exhausted after the first 24 hours growth; after this point, the cells enter the lipid
accumulation phase in which the excess carbon (e.g., glucose) continues to be assimilated by
the cells, and because there is no new cell synthesis because of the lack of nitrogen, the surplus
carbon is converted into lipid, which functions as a reserve of carbon and energy for the cells.
129
Under the conditions of nitrogen-limited growth, the first requirement is for the
cells to cease generating energy (i.e., ATP), which is no longer needed for the
synthesis of new macromolecules, (e.g., proteins and nucleic acids) as the cells
are unable to grow and divide because of the lack of nitrogen (required for protein
biosynthesis). A key enzyme in the citric acid cycle (Krebs cycle or tricarboxylic
acid cycle), isocitrate dehydrogenase, becomes inactive immediately following the
depletion of nitrogen. This leads to isocitrate not being metabolized, and consequently, both it and citric acid, with which it is in equilibrium, rapidly accumulate.
This event occurs in the mitochondria of the cells, but the citrate is quickly transported out of this compartment into the cytoplasm of the oleaginous cell and is
immediately cleaved by an enzyme known as ATP:citrate lyase (1):
Citrate ATP coenzyme A ! acetyl-coenzyme A oxaloacetate ADP
inorganic phosphate . . . . . . . . . . . . . . .
This is a key reaction as it generates the C2 building unit (acetyl-CoA) for fatty acid
biosynthesis. Without this enzyme being present, there would be no abundant supply of the acetyl-CoA units and, indeed, many if not all of the nonoleaginous yeasts
do not possess this enzyme. The oxaloacetate generated in this cleavage reaction is
immediately converted to malate by malate dehydrogenase and then the malate, in
turn, is converted to pyruvate by the action of malic enzyme (2).
Malate NADP !pyruvate CO2 NADPH . . . . . . . . . . . .
This is the second key enzyme needed to produce high amounts of lipid as the reaction catalysed simultaneously produces the necessary reducing equivalent, NADPH,
by which the growing long acyl chain, derived from acetyl-coenzyme A (see
above), is reduced to the final long-chain fatty acid. Fatty acid biosynthesis, and
consequently lipid accumulation, requires both a continous supply of acetyl-CoA
and reducing power (NADPH), and these are provided by the key reactions mentioned above.
How the oleaginous microorganism then produces variable amounts of lipid (see
Table 1) lies with the activity of malic enzyme rather than in ATP:citrate lyase.
Malic enzyme activity is controlled by the genetic makeup of the cell: in cells
that accumulate considerable amounts of lipid (up to and even above 70%), the
gene that controls the synthesis of the malic enzyme is kept switched on all the
time, whereas in the low-lipid cells, the gene is switched off shortly after nitrogen
exhaustion. When this happens, malic enzyme activity quickly disappears and, concomitantly, lipid accumulation ceases. Thus, it is now possible to explain not only
the reason why some microorganisms are able to accumulate lipid and other cannot,
but also why the amount of lipid accumulated can vary considerably within the
lipid-accumulating organisms (8). This biochemical information is now leading to
the identification of the key genetic elements that are involved in lipid biosynthesis.
From this will then come the opportunity to genetically modify microorganisms
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to give them increased quantities of lipid and, simultaneously, to produce more of

the desired fatty acids.
2. COMMERCIAL MICROBIAL OILS

2.1. Initial Ventures Into Single Cell Oil Production
Although microbial lipids,
SCO, were considered as possible commercial sources of oils and fats for almost
all of the last century, no industrial production of a microbial oil took place
simply because no economically attractive target had been identified. However,
by the late 1970s, certain polyunsaturated fatty acids were being used medicinally as
over-the-counter treatments for a variety of disorders, chief among which was the
use of evening primrose oil (9) as a possible treatment for multiple sclerosis.
Evening primrose oil contains the relatively unusual fatty acid, gamma-linolenic
acid, 18:3n-6. (GLA) (see Table 2). Claims for the efficacy of this oil in the treatment of many disorders, including atopic eczema (10, 11), which go back many
centuries in a historical context, are then attributed to the presence of this fatty
acid, which does not occur in most other plant seed oils. Other claims for the efficacy of GLA-rich oils include the treatment of premenstrual tension, which is the
main selling point of this oil in the United Kingdom, as well as for various cancers
(9, 12). These have added impetus to identifying a cheaper and perhaps more reliable source of GLA as the cultivation of evening primrose is not easy, with it being
a biennial crop and which produces very tiny seeds requiring careful harvesting and
processing (13). As it was known that GLA is also present in a large number of
simple fungi, known as the lower fungi or Zygomycetes since the 1940s, a fungal
route to its production was recognized as a feasible alternative to evening primrose
2.1.1. Fungal Oils Rich in Gamma-Linolenic Acid
TABLE 2. Fatty Acid Profiles of GLA-SCOsMicrobial Oils Rich in Gamma-Linolenic

Acid (18:3 n-6)in Comparison with Plant Sources of GLA [Adapted from (4) and 6)].
Relative% (w/w) of Major Fatty Acids
Oil
Content 16:0
(% w/w)
Mucor circinelloides1
Mortierella isabellina2
Mortierella ramanniana
Mucor hiemalis
Evening primrose
Borage
Blackcurrant
1
25
ND
40
30
16
30
30
22
27
24
25
6
10
6
16:1
18:0
18:1
18:2
(n-6)
1
1
6
6
5
10
2
4
1
40
44
51
32
8
16
10
11
12
10
12
75
40
48
Production organism used by J & E Sturge Ltd., Selby, N. Yorks., UK.

Production organism used by Idemitzu Ltd., Japan.
NDNot disclosed but believed to be 40% to 50%.
2
18:3 18:3 20:1 22:1

(n-6) (n-3)
18
8
10
15
8
22
17
0.2
0.5
13
0.4
0.2
4.5
2.5
COMMERCIAL MICROBIAL OILS
131
oil. Work was carried out in the authors laboratory at the University of Hull beginning in 1976 by screening a large number of these fungi for their potential to produce an oil rich in this fatty acid. This led to Mucor circinelloides (formerly known
as Mucor javanicus) being identified as the most productive organism (3): although
this fungus was not the highest producer of GLA, there were three important and
interdependent variables that had to be satisfied:
The organism had to be able to grow quickly and to a high cell density. A
working target was for an organism to achieve more than 50-g dry cell per
liter of fermenter in a time not to exceed 4 days.
The organism had to have more than 20% oil content; otherwise extraction
would be difficult, and the costs of oil production would be increased. Equally,
the extracted oil should be at least 90% as a triacylglycerol so that subsequent
refinement and encapsulation would be relatively easy to accomplish.
The content of GLA in the oil had to be considerably higher than that in
evening primrose oil, which was only about 10%; a working target of 20%
was therefore chosen.
A fourth preference was for the chosen organism to be already recognized as
being safe to use for food purposes: the so-called Generally Recognised As
Safe (GRAS) category. However, this was not regarded as absolutely essential
as the product would be an extracted, purified oil. It would then be the quality
and safety of this that would be assessed rather than the safety of the whole
organism. Nevertheless, one would not want to use a microorganism that had
any association with any disease for very obvious reasons. Also, the chosen
organism should not be a plant pathogen because of possible environmental
risks when growing the organism on a large scale. (All organisms being used
in large-scale cultivations have to be intrinsically safe to handle and even
organisms that could cause allergic reactions in factory operatives are best
avoided.)
The finally chosen production organism, Mucor circinelloides, satisfied all of the
above criteria, including it being a GRAS-status organism as it has a historically
long association with tempe, a well-known oriental food material. The profile of
its fatty acids is given in Table 2 along with its lipid content.
In Japan, Idemitzu Co. Ltd. adopted a slightly different strategy to isolate GLAproducing organisms. They opted to go primarily for organisms producing high oil
contents and, seemingly, hoped that the GLA also would be high in such organisms.
In the event, although high oil-producing species were found, none produced more
than 10% of the total fatty acids as GLA (see Table 2). For a reason not yet understood, but possibly related to the limited capacity of the cells to generate NADPH
that is used both in fatty acid synthesis and in fatty acid desaturation (see Section
1.3), there is an inverse relationship between oil content and GLA formation. The
higher the oil content of the cells, the lower is the GLA content (see Table 2). The
Idemitzu oil from Mortierella isabellina, therefore, had only half the GLA content
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of the U.K. organism, Mucor circinelloides, although the Japanese organism had
twice the oil content of the U.K. organism. Commercially, however, the higher
the GLA content of an oil, the greater its value, and an oil with 20% GLA is probably more than twice as valuable as an oil with only 10% GLA, so it is more costeffective to produce more GLA at the expense of producing less oil.
The GLA-SCO was produced in the United Kingdom by John & E. Sturge,
Selby, North Yorkshire, using Mucor circinelloides. It was first offered for sale in
1985 under the name of Oil of Javanicus, which took account of the oriental origins
of the organism and its original name, Mucor javanicus. The oil was sold commercially throughout the United Kingdom between 1985 and 1990. Production
was at about 1015 tons of oil per year. The fermenters used were 220,000 L
and were normally used for the production of citric acid using Aspergillus
niger. The overall fermentation configuration was similar to that shown in
Figure 1. To grow M. circinelloides for oil production, rather than A. niger for citric
acid, all that had to be done was to reformulate the growth medium so that it
now contained an insufficient supply of nitrogen and so that cell proliferation
would cease after about 3640 hours and lipid accumulation would then begin
(see Figure 2).
The Oil of Javanicus was of high purity and passed all toxicity tests: It was
superior in safety evaluations to conventional plant oils, which always contain
very small residual amounts of pesticides, insecticides, and herbicides from the various sprays that are used on such crops. These levels, though, are always below the
recommended threshold values laid down by regulatory authorities. Being cultivated in fermenters, M. circinelloides does not, of course, need to be sprayed
with any chemical pesticide, herbicide, or fungicide.
The arrival of this novel oil on the market resulted in a sharp decline in the price
that evening primrose oil was being offered for sale. Competition between it and
Oil of Javanicus was much fiercer than had been anticipated. Although for all
intents and purposes, the fungal oil was superior to the plant oil, in that it contained
twice the GLA content of the evening primrose oil, nevertheless, and perhaps not
surprisingly, there was a certain reluctance on behalf of the general public, who
were buying these oils to switch to an oil of fungal origin. However, the marketing
of the oil carefully eschewed specific mention of the word microorganism or
fungus, but nevertheless there was a reluctance on the behalf of major purchasers, the health-food stores, over-the-counter medicine shops, and so on, to purchase the oil in spite of its technical superiority. Although interest in this GLArich oil was high, it quickly became apparent that it was being outcompeted by evening primrose oil in terms of price. Unfortunately, within the European Union, agricultural crops not designated as food crops could be financially subsidized from the
Common Agricultural Policy. This meant that growers of evening primrose oil
received cash benefits directly from the EU for growing this plant, which was designated as a non-food crop. At the same time, the fermentation process was being
financially penalized by the sugar used as the feedstock had to be bought within
the EU at EU prices and not at world prices, which were less than half this cost.
Sugar within the EU has a tariff placed on it so that farmers in the EU can receive
133
adequate remuneration for growing this crop. Thus, the Oil of Javanicus was doubly
disadvantaged by its commercial rival, evening subsidized primrose oil, which was
subsidized by the EU, and at the same time the cost of production was increased by
the artificially high price of the fermentation feedstock.
The final blow to the production of the fungal oil came with the introduction of
borage oil (Borago officinalis) as a superior source of GLA. This new oil had a
GLA content of 2022% (see Table 2) and, although it was technically just as difficult to grow and process as evening primrose oil, it was considered superior and
was, of course, cheaper, than both the evening primrose oil and the fungal oil.
Again, growing this borage crop still enjoyed the financial benefits that had accrued
from the EU Common Agriculture Policy for evening primrose cultivation.
In 1990, and against this background of increasing erosion of the profit for the
microbial SCO, production of Oil of Javanicus ceased. The 6 years in which the oil
had been in production, though, established a number of important points. First,
microbial oils could be produced on the very largest of scales, up to 220 m3 in
this case. The oil was intrinsically safe, posed no safety problems, and passed all
toxicity trials to which it was subjected. It was well accepted by all people who
consumed it, and no adverse reactions to it were ever recorded. There were no
particular difficulties in extracting the oil (a process using hexane was used), and
the oil could be easily purified using conventional procedures of the oils and fats
industry. The oil was also remarkably stable from oxidation, presumably because of
the presence of endogenous natural antioxidants. Thus, the way was now open for
other microbial oils to enter the market even though Oil of Javanicus was no longer
in commercial production.
In Japan, the GLA-SCO from the Idemitzu Co. Ltd process using Mort. isabellina appears also to have ceased production, and GLA, when needed, is derived
either from evening primrose oil or borage oil.
2.1.2. Cocoa-Butter Equivalent Yeast Fat The initial interest in producing
high-value microbial oils in the late 1970s quickly led to serious consideration
being given to the prospects of producing a facsimile oil to cocoa butter, i.e., cocoa
butter equivalent fat or CBE (1416). The price of cocoa butter, which is used
extensively in chocolate manufacture, is very variable, but in the early 1980s, its
price was exceeding $8000 per ton. CBEs are traditionally made by palm oil fractionation to give an oil containing a high content (approximately 3035%) of stearic
acid (see Table 3) with equal proportions of oleic acid and palmitic acid (17) and
command a price that is usually fixed at about 50% to 60% of the price of cocoa
butter. CBEs can be added into cocoa butter at up to 5% in various countries,
including the United Kingdom, without invalidating the claim for the product to
be called chocolate. In other countries, such mixtures can only be used in confectionery chocolate.
The high price of cocoa butter at this time then made it an attractive target to
emulate. A sufficient margin of profit was considered possible if a microbial oil
could be produced that mimicked the fatty acid profile of cocoa butter and, most
importantly, had the same melting profile as cocoa butter. This manifests itself
134
TABLE 3. Fatty Acid Profiles of CBE-SCOsMicrobial Oils for use as a Cocoa Butter
Equivalentin Comparison with Cocoa Butter.
Relative % (w/w) of Major Fatty Acids
Cryptococcus curvatus WT a
C. curvatus NZ
C. curvatus R26-20
C. curvatus R25-75
C. curvatus F33.10
Trichosporon cutaneum DRL-D221
T. cutaneum DRL-ole
Yeast isolate K7-2
Cocoa butter
16:0
18:0
18:1
18:2
30
18
15
33
24
30
26
26
15
24
47
25
31
13
38
25
45
48
25
33
30
47
16
38
5
3
8
7
6
32-37
30-37
2-4
23-30
18:3
(n-3)
24:0
Ref
0.5
1
2
1
7
14
1
2
2
27
28
21, 23
21, 23
22, 23
24
24
29
WT wild type.
by the fat being solid at ambient temperature (up to about 25 C) but then melting
completely at 3032 C. As most microbial oils contain less than 10% of the total
fatty acids as stearic acid (18:0), the task was then to increase the content of stearate
and, simultaneously, to ensure that the resulting triacylglycerol had the correct fatty
acid distribution (i.e., was a sn-1 palmitoyl, sn-2 oleoyl, sn-3 stearoyl glycerol) so
that the ensuing oil would meet the very stringent requirement for its inclusion in
chocolate.
The most attractive production organisms appeared to be yeasts, which do not
usually contain high amounts of 18:2 or other polyunsaturated fatty acids and therefore were immediately attractive for this reason. They also could be grown extremely rapidly and to high cell densities with high lipid contents. Attempts in the
early 1980s had been made by several groups to increase the amount of stearic
acid in a yeast by growing the yeast on stearic acid, or esters of stearic acid, as feedstocks (5). Not surprising, such yeasts then contained high amounts of this acid, but
as no cheap source of the stearic acid existed at this time, this did not represent an
economic route to an SCO-CBE product.
An alternative strategy was developed by Cadbury-Schweppes plc, the large
U.K.-based, multinational chocolate and food company, to inhibit the conversion
of stearic acid to oleic acid, which is mediated by fatty acid delta-9 desaturase,
using sterculic acid, cis-9,10-methyleneoctadecenoic acid (18, 19). This inhibitor,
which can be derived from sterculia and kapok oils (18), increased the stearic acid
content of several oleaginous yeasts up to nearly 50% of the total fatty acids when
they were grown on glucose. However, the selectivity of the inhibitor was such that
the yeast still contained too much linoleic acid, and consequently, another inhibitor
of the delta-12 desaturase (converting oleic acid to linoleic acid) was needed.
This was cis-12,13-methyleneoctadecenoic acid, which had to be synthesized
135
chemically. In the presence of both inhibitors at about 100 mg/L, one yeast,
Rhodosporidium toruloides, now produced a SCO-CBE product that was close
to the required fatty acid profile (5, 19). The costs of the fatty acid desaturase
inhibitors unfortunately proved to be too high for the process to be sustained,
and there was also clear unease at using metabolic inhibitors that might find their
way into the final product. Consequently, this interesting and novel approach was
abandoned.
Nevertheless, the clear conclusion was reached from the work with sterculic acid
that an SCO-CBE was possible if the activity of the delta-9 (and the delta-12) desaturase could be diminished. This was then the concept behind the subsequent mutation program that was developed by several groups but most notably by a group at
the Free University of Amsterdam, The Netherlands. The yeast chosen for this
mutational work was Candida curvata (also known as Apiotrichum curvatum and
now renamed Cryptococcus curvatus). Using conventional mutational procedures,
various mutants were produced that had lost the ability to synthesize oleic acid and
now needed this fatty acid to be included in the growth medium (2023) (see also
Table 3). Clearly the gene coding for the delta-9 desaturase had been affected by the
mutations.
By judicious selection of the various mutants and adjustment of the mutational
makeup, it was possible to select mutants that had a low activity of the delta-9 desaturase and no longer needed oleic acid to be added as a supplement to the growth
medium. The composition of the fatty acids of two of these mutants, R26-20 and
R25-75, are shown in Table 3. The best results were, obtained though, using a
hydrid of two mutants, F33.10, which gave an almost ideal fatty acid profile for
an SCO-CBE (see Table 3). Similar mutational programs were carried out by other
research groups (2426), although with no greater success than the Dutch group
(23).
One factor that is a prerequisite for using mutated strains of micro-organisms is
their long-term genetic stability and, in particular, their stability when grown in
large-scale fermenters where they have to undergo many generations to reach the
required cell densities. Also, it is important that the mutants should grow as rapidly
as the original parent organism. During the growth of the mutants from small cultures (say, 200 mL) up to the final level at perhaps 100,000 L or higher (see Figure 3),
there has to be complete genetic stability; otherwise the organism reverts to its original constitution and the desired product is no longer produced. For these reasons,
the mutants of the yeast used for SCO-CBE production were not entirely dependable when used in large-scale growth trials and yields of the required CBE were
below expectation. This is probably attributed to there being other mutational
changes to the DNA, besides the alteration in the oleic acid desaturase gene, which
then affect the long-term performance of the organism. Today, current molecular
techniques would allow one to identify the key genes that needed to be changed
and then these could be individually manipulated in such a way that the remainder
of the DNA would not be affected. Thus, in principle, specifically, genetically modified yeasts could now be produced that would yield a very high-quality SCO-CBE
even when grown in large-scale fermenters. However, in the 1980s, before this
136
current technology was available, further efforts to produce a yeast oil CBE focused
on controlling the production of oleic acid using a restricted supply of oxygen to
the cells.
As the conversion of stearic acid to oleic acid (and indeed all desaturase reactions) requires the active participation of O2 in the reaction, it was attractive to consider that the content of stearic acid in the yeast oil could be increased by restricting
the supply of air to the growing cultures. This, in fact, was achieved by Davies et al.
(28) using the yeast, C. curvatus. To achieve the necessary low levels of oxygen
within the fermenter, a 500-L system had to be employed. With smaller fermenters,
it was not feasible to restrict the oxygen supply sufficiently to cause the desired
effects. The fatty acid profile of the yeast when grown under such conditions is
shown in Table 3 and was considered to be a reasonable approximation to that
needed for the oil to be considered as a suitable CBE (27).
Extensive large-scale cultivation of various yeasts, but principally C. curvatus,
for the production of SCO-CBE was carried out by Davies et al. in New Zealand
from the early 1980s until the 1990s. The concept behind this work was the use of
cheese creamery waste, which in New Zealandthe location of Daviess laboratorywas a major waste resource. As whey contains 4% to 5%(w/v) lactose,
this could be used as an appropriate substrate for yeast growth. The yeasts chosen
by Davies (see 29), therefore, were all lactose-users, including C. curvatus, which
had originally been isolated by Earl Hammond et al. from dairy waste-processing
areas (30). By virtue of the extensive work carried out on this process, Davies et al.
was able to calculate the likely costs of production of an SCO-CBE (27, 29).
Davies, writing in 1992 (27), calculated that the manufacturing cost of producing
1 ton of refined yeast oil would be $800$1000. This was based on a process
that would use 200,000 m3 of whey per year and that would be available at a single
location in New Zealand. These costs, though, did not include plant depreciation,
the interest payable on the capital investment needed, nor the manufacturing overheads. Collectively, these costs could then double the original estimate. At this time
(late 2003), cocoa butter had slumped in world prices from its all-time high of
$8000/ton in the early 1980s to $3500/ton. As a CBE could only command about
two-thirds of this price, this meant that the yeast CBE would only be valued at
$2000$2500/ton. The margin of profit, therefore, was not great enough to warrant
the large investment of capital that would be needed to establish the process.
Consequently, after huge efforts by many groups around the world, it was generally
agreed that this approach was not economically viable. As far as the present authors
are aware, no further commercial interest has been shown in developing a
yeast-based process for the production of a CBE. Even if the economics of cocoa
butter production were to change in the next decade, there are now sufficiently
good alternative systems, including the use of immobilized lipase technology, to
produce CBE material more cost-effectively than the microbial route of manufacture (17, 31). It is worth pointing out, however, that the cost-calculations of Davies
for the bioengineering aspects of SCO-CBE production could form the basis for
future calculations of such processes aimed at the production of other microbial
oils.
SCOS IN CURRENT (2003) PRODUCTION
137
3. SCOS IN CURRENT (2003) PRODUCTION

As already discussed, the cost of SCO manufacture is high because of the high capital costs involved in the construction of large fermenters and associated machinery
as well as in the costs of operation. If a microbial oil is to be exploited commercially, then the SCO must command a premium price (32, 33). In reality, this means
that microbial sources for oils can only be a commercial reality if the SCO produced is (1) destined for human consumption and (2) not readily available from
traditional sources, either plant or animal.
Such opportunities exist in terms of long-chain polyunsaturated fatty acid (LCPUFA) rich oils that have well-defined and publicly recognized benefits for human
health. Currently, >95% of the global SCO production are oils rich in two distinct
fatty acids:: arachidonic acid (ARA, 20:4 n-6) and docosahexanoic acid (DHA,
22:6 n-3). Both are being produced either directly by, or under contract, for Martek
Biosciences Corporation, MD. The production of SCOs is no longer a small-scale
operation. The blend of SCO produced by Martek as a neonate nutritional supplement (for inclusion in infant formula) has established itself worldwide in the past
few years, to the extent that current production is measured in the hundreds of
tons annually. An estimate has been made that for 2003, about 560 tons of SCOs
will be produced, the majority of which will be SCOs rich in either ARA or DHA.
For 2004, this amount is predicted to double and possibly double again in 2005.
ARA and DHA are LC-PUFA, having a carbon chain of greater than 18 carbons
and are not available from plant sources. Agricultural plants usually produce
only 18 carbon (or shorter) fatty acids, and hence, the most unsaturated fatty acids
they produce are 18:3 of the n-3 or n-6 type. Although some plants, though, do produce C20 fatty acids, these are monounsaturated such as erucic acid (22:1, n-9) or
nervonic acid (24:1, n-9) and do not possess the beneficial physiological effects of
ARA and DHA. Animals (including fish) are potential sources of a multitude of
polyunsaturated fatty acids with carbon chains as long as 22 and as many as six
double bonds. However, these animal oils have potential problems of generally
low LC-PUFA content and complex fatty acid profiles, as well as acceptability problems to various sectors of the community based on religion or lifestyle (34).
Furthermore, there are increasing concerns about the presence of viruses and prions
in materials of animal origin, and with fish, there are worries concerning their longterm availability as a cheap resource. More recently, reports of the possible accumulation of toxic pollutants, including heavy metals, from the marine environment
into fish livers have added another dimension to the argument against the use of fish
oils. Fish livers, together with whole fish bodies, are, of course, the current major
source of LC-PUFAs.
SCOs have the advantage over these traditional sources for the provision of LCPUFA for the very reason that they are produced by fermentation. The quality and
supply of SCO can be closely controlled and guaranteed on a yearly basis (32, 33).
Such guarantees are hard to provide for plant-or animal-derived oils because of
environmental conditions and variations that are outside of the producers control.
Weather, diet (for animal oils), and environmental pollution (including the spraying
138
of almost all commercially grown crops with mixtures of herbicides, pesticides, and
fungicidal chemicals) have a considerable impact on the supply and quality of oils
from traditional sources. Also, the growth of micro-organisms is very rapid in comparison with agricultural crops and animals; usually fermentation times are 4 to 10
days. As a result, the supply of SCO can far more easily be matched to the market
requirement, avoiding oversupply or shortage problems that can be an issue with
traditional oils.
Initially, public reluctance to consume a microbe-derived oil in preference to
those from plant or animal sources was a considerable problem, contributing to
the decline of Oil of Javanicus (the first commercially produced SCO); see Section 2.1.1. However, a mixture of more astute marketing, rebranding of SCO as
either fermented oils, designer oils, or even vegetarian oils when in competition with animal oils, and the improved public acceptance of microbial
foods, as exemplified by the success of the mycoprotein (SCP) product, QuornTM,
in the United Kingdom and Europe, has helped establish PUFA-SCOs as acceptable
products.
3.1. Archidonic Acid
After the success (at least temporarily) of Oil of Javanicus, the next fatty acid to
be developed on a commercial basis was ARA. The early development occurred in
the Far East, with two Japanese companies, Lion Corp. and Suntory. Both companies developed processes for producing arachidonic acid using fungal sources. It is
interesting to note that the interest of Lion Corp. in an arachidonic acid-rich oil was
not for nutritional applications but as the basis of cosmetic creams, another area that
can withstand commodities with a premium price. This development resulted in
patents being awarded to these two companies in the late 1980s covering the production of arachidonic acid from Mortierella alpina (35, 36).
ARA-rich SCO continues to be produced commercially in the Far East by Suntory and is produced in Europe by Dutch State Mines (DSM), formerly Gistbrocades. DSM produces ARASCOTM (an oil produced from the fungus Mortierella
alpina and containing >40% w/w ARA) under contract for Martek Biosciences
(USA) for inclusion in the neonate Formulaid1 nutritional supplement, which
is used in infant formulas and baby foods in 60 countries worldwide (including
the United States since February 2002). Currently, infant formula is, by far, the
most important market for SCO. Over 95% of global sales of SCO is made up
by a single product, Marteks Formulaid1 (Figure 4).
The astonishing success of this product is evidenced by the fact that although it
was introduced into infant formula in the United States in February 2002 (and the
formula containing this supplement commands a premium price), by the time of
writing (September 2003), the market share of infant formula containing Formulaid1 in the United States has reached 30%. The ARA-rich SCO produced by Suntory, which is similarly used in infant formula, is mainly for export as the Japanese
market for such products is still being developed. However, the oil is available within Japan as a health supplement. In China, the Wuhan Alking Bioengineering Co.
139
Figure 4. The production and uses of commercial SCO over the past 20 years and comparison
with current estimated production and use (Dr. David J. Kyle, personal communication). (This
figure is available in full color at http://www.mrw.interscience.wiley.com/biofp.)
Ltd., located in Wuhan City, has been producing arachidonic acid using Mort. alpina since 2001 (see www.alking.com.cn). Production appears to be at the 50,000-L
level although larger fermenters may be under commission. The oil is thought to be
used in infant milk powder but also may be exported to unknown destinations.
As Mort alpina, unlike Mucor circinelloides (also known as Mucor javanicus),
did not, prior to the production of ARA-rich SCO, have any definite historical association to any food product, extensive toxicological studies were performed to confirm the safety of ARASCO (3740). This requirement for toxicological screening
is a hurdle that must be overcome for any SCO that cannot claim GRAS status by
association with a preexisting human food. Obtaining this toxicological data
ab initio can be a costly and time-consuming process.
Many micro-organisms have been examined for the ability to produce substantial
amounts of ARA, but the overwhelming consensus is that fungi of the genus
Mortierella (and in particular the subgenus of Mortierella Mortierella) are the organisms of choice (41 43). Although a number of species of Mortierella have been
suggested, and even perhaps developed, as production organisms (44), the commercial production of ARASCOTM is currently carried out using Mortierella alpina.
The major culture collections ATCC (14 strains), IMI (4 strains), CBS (19 strains),
and so on, contain multiple isolates of Mortierella alpina, which have been exhaustively examined for their commercial viability. The most productive strain will
depend to a large degree on the process developed as strain selection, and process
development must occur in parallel. The interdependence of the strain/process complicates the isolation/selection of new improved strains once a process is established.
140
The most productive strain in the open access culture collections appears to
be ATCC 32222 (45, 46). However, the group of Shimizu working in collaboration
with Suntory in Japan have isolated a very high ARA-producing environmental
strain that has been designated Mortierella alpina 1S-4 (32, 36, 41). This strain
has several unusual and advantageous characteristics (ease of sporulation amongst
them). Much experimental data pertaining to this strain exist in the literature (32,
47 49). However, because of the proprietary nature of this strain, little of this published data has had the opportunity to be corroborated by other workers in the field.
Although relatively high-producing strains of Mortierella alpina are available
off the shelf from culture collections, it is inevitable that commercial production
will use proprietary improved strains. The processes used to obtain these strains
involve a certain amount of lore because of the difficulties obtaining spores of
the organism, which are a prerequisite for carrying out a successful mutation
program. Also, as increased ARA/oil production within a mutant organism confers
no easily recognizable trait in a mutant strain, considerable effort must be expended
to screen many thousands of potential mutants to isolate one new strain that overproduces ARA.
The commercial production of ARA-rich SCO
starts with the thawing of a cryovial of a certified stock culture of the production
strain of Mort. alpina. The thawed cryovial is used to inoculate a series of shakeflask cultures and seed fermenters of increasing volume to maintain a 510% inoculum for each successive stage of the seed train. Final production scale is typically
50 to 200 m3 (see Figure 2) (50).
Media used for ARA-rich SCO production vary depending on the process/strain
used; however, they tend to be relatively simple complex media composed of a base
of yeast extract and glucose (51), although the ion composition is known to be crucial for optimal productivity as well as the carbon to nitrogen ratio in the medium
(48, 52, 53).
Unlike classic fungal fermentations, such as for the production of penicillin,
citric acid, and gibberellic acid (1), the desired product in the case of ARA-rich
SCO is an intracellular product and this leads to the ARA-rich SCO process having
some distinctive features. Although in the other processes fungal biomass is, in
effect, an unwanted byproduct and can be minimized, in the ARA-rich SCO process, a very high cell density must be achieved. Cell densities as high as 5060 g/lL
dry weight have been reported (48), and this creates problems in terms of culture
mixing and mass transfer as LC-PUFA biosynthesis is an arobic process caused by
the O2-requirement of the fatty acid desaturases.
In order to obtain maximal ARA-rich SCO productivity, the fungus must be cultivated in the correct morphological form. Mort. alpina can grow as either dispersed
hyphal filaments or as pellets (of varying sizes; depending on conditions). Although
feather-like hyphal filaments yield the optimal ARA production at low cell densities, because of the ready access of nutrients and ease of gas exchange between
the hyphae and the medium (54), this morphology is not suitable for cultivation at
high cell densities. Under intensive cultivation conditions, hyphal growth causes the
3.1.1. Commercial Production
141
viscosity of the culture to increase and agitation of the culture to be difficult (48).
Under these conditions, gas exchange and nutrients become limited, thereby deleteriously affecting ARASCO production. For large-scale industrial ARA-rich SCO
production, the optimal morphology is pellets, which causes a decrease in the viscosity of the culture and promotes mixing. When pellets form, however, the culture
becomes heterogeneous as the biomass in the interior of the pellet can experience
nutrient limitation (49). Depending on the size of the pellets, the ARA-rich SCO
production when the fungus changes from hyphal to pellets can be decreased by
as much as 50% (45). Therefore, achieving the optimum morphology for ARArich SCO production is not a trivial matter. It appears small pellets < 2 mm in diameter are optimal to decrease culture viscosity, while maintaining adequate nutrient
and oxygen transfer to the pellet interior (48, 55).
The ARA-rich SCO fermentation operates under nitrogen-limiting conditions to
promote cell lipid accumulation. A glucose-fed batch system is employed to allow
high levels of carbon to be used without the toxic effects of very high initial glucose
concentrations. Once the biomass has accumulated a suitable amount of lipid and
ARA (approximately 200 mg ARA/g dry weight), the fungal biomass must be
removed from the culture medium, either by continuous centrifugation or filter
pressing, and dried before the oil can be extracted and purified using techniques
essentially identical to those used for vegetable oil production. The resulting oil is
a pale yellow brilliant oil with a relatively bland flavor that is remarkably resistant to
oxidation. The fatty acid profile of the commercial oil is given in Table 4 (55).
3.2. Docosahexaenoic Acid
The most unsaturated fatty acid found in significant quantities in nature (some algal
lipids contain trace amounts of fatty acids with as many as eight double bonds) is
DHA. This fatty acid is known to be especially important in the neural development
in animals; indeed, DHA alongside ARA are the predominant PUFA in neural
tissue (56). A deficiency in DHA in has been linked to impaired brain and visual
development in neonates (56).
Once the physiological role of DHA had been realized, this fatty acid became an
obvious candidate for commercial microbial production as no traditional lipid
source rich in this LC-PUFA alone was known. As mentioned above, plants generally do not synthesize fatty acids longer than 18 carbons and no plant makes any
DHA. DHA is found in animal-derived oils, particularly fish oils; however, the use
of fish oil as a DHA supplement is problematic. Fish, like all animals, possess fatty
acid profiles that, to a large extent, reflect their dietary intake rather than metabolic
ability to synthesize fatty acids. As a result, the fatty acid profiles of fish oil are
complex and contain a host of other PUFA as well as DHA. The presence of another
LC-PUFA, eicosapentaenoic acid (EPA, 20:5 n-3), is a particular problem associated with fish oil if the intended use is neonate nutrition. EPA, a precursor of
important signal molecules (prostaglandins and eicosanoids), is thought to act
antagonistically to the beneficial effects of ARA-rich SCO supplementation of
infant formula and is associated with growth retardation in neonates (57).
TABLE 4. Fatty Acid Profiles of Microbial Oils Rich in Arachidonic Acid and Docosahexaenoic Acid that are Produced Commercially (from 55).
Fatty Acid Composition (Rel. % w/w)
12:0
14:0
16:0
16:1
8
18
29
0
2
12
18:0
18:1
18:2
(n-6)
18:3
(n-6)
18:3
(n-3)
20:3
(n-6)
20:4
(n-6)
22:5
(n-6)
22:6
(n-3)
24:0
14
15
1
7
0.6
2
49
12
0
39
25
Oil
ARASCOa
DHASCOb
Schizo-SCOc
a
0.4
20
13
11
0.4
1
Production organism: Mortierella alpina (see 55).

Production organism: Crypthecodinium cohnii (see 55).
Production organism: Schizochytrium sp. (see 55).
NB: ARASCO and DHASCO are registered tradenames of Martek Corp. Inc.
b
c
143
Other problems associated with the administration of fish oil to the general population, and in particular to neonates, is the potential for the presence of environmental toxins in fish oils. As the fish from which the oils are derived are free living in
the worlds oceans, they are prone to contamination with pesticide residues and
industrial wastes (including heavy metals) that are all too frequently released
into the marine environment. Although the levels of these chemicals are usually
low, they are of sufficient amounts to cause concern that the British Government
commissioned a report that has highlighted the extent of contamination of fish
stocks (58). In particular, the presence in fish oils of dioxins and polychlorinated
biphenyls (PCBs) was noted and the report recommended that oily fish should
not be eaten more than once a week. More recently, the specter of mercury in
fish oils has led to the recommendation by the Food and Drug Administration
(FDA) and the Environmental Protection Agency in America (59) that pregnant
women should restrict their oily fish intake. Furthermore, a recent study has determined that the mercury content of fish oil diminishes the cardiovascular benefits of
these oils (60). Any inclusion of the oils from such fish, mainly derived from liver,
an organ that often concentrates ingested toxins in infant formula, even if the levels
of contaminants is low and less than safety limits, must be seen as foolhardy. Alternative sources of DHA that would be outside the current supplies are clearly
needed, and such sources may then be expected to command a premium price
far in excess of that of fish oils.
3.2.1. Microbial Sources of DHA It has been recognized for over 30 years that
certain marine microorganisms have the ability to synthesize DHA de novo and to
accumulate significant amounts of this fatty acid in their cellular lipid in a relatively
pure form (61). Indeed, it is these microbes at the base of the food chain that
synthesize the DHA that eventually appears in fish oil (62). Two microalgal
sources, in particular one dinoflagellate (Crypthecodinium cohnii) and one chytrid
(Schizochytrium), have been found to synthesize sufficient quantities of DHA (in an
oil devoid of EPA) to become commercially viable. Although both organisms produce a DHA-rich SCO, important differences exist in the fatty acid profiles of the
resultant oils that have significant impact on their potential use. Although C. cohnii
oil contains DHA as >99% of the PUFA (and >40% of the total fatty acids), the oil
from Schizochytrium contains a significant amount of another PUFA docosapentaenoic acid (DPA n-6; 22:5n-6). The presence of DPA in the chytrid oil was initially a
cause for sufficient concern that this oil was not considered suitable for neonate
nutritional applications. However, the DPA appears to be completely benign and
has, moreover, been recognized as a natural component of the phospholipids of
human blood platelets (63, 64). Studies of DPA metabolism in rat hepatocytes
(65) have further indicated that DPA is retroconverted to ARA if the ARA content
of the diet is low, but when ARA is administered along with a mixture of DPA and
DHA, then the DPA serves to maintain the DHA at a high circulating concentration.
Thus, there may be positive benefits of including DPA in a dietary supplement of
ARA DHA, and this would occur by using the Schizochytrium oil along with the
ARA-SCO.
144
Initially, the two organisms were developed as competing processes by two pioneering U.S. firms, Martek (using C. cohnii to produce DHASCOTM, an oil containing >40% DHA) and OmegaTech (using Schizochytrium to produce Sea GoldTM,
now known as DHAGoldTM, an DHA-rich SCO also containing DPA n-6). In April
2002, Martek acquired OmegaTech to combine the expertise of the two companies.
Currently, the DHA-rich SCO produced by Martek is almost exclusively DHASCOTM and is destined for infant formula as part of the SCO blend Formulaid1.
The production of the Schizochytrium oil continues at a low level and is aimed
more directly at food applications. Several other companies, Norferm in Norway
and Celanese in Germany, have developed competing processes to some level at
least, and indeed Celanese, through their subsidiary company Nutrinova, Frankfurt,
Germany, have recently expressed their intent to launch a chytrid-derived DHA-rich
oil, to be known as DHActiveTM. To date, however, no DHA-rich SCO other than
Marteks DHASCOTM has reached a large-scale market. In Japan, Nagase-Suntory
are thought to be producing small amounts of DHA-SCO to be used as a dietary
supplement, although it is uncertain to what extent production is taking place.
Both C. cohnii and Schizochytrium were selected for both their capacity to accumulate large amounts of DHA containing oil (cells contain >30% oil containing
>40% DHA) and ease of cultivation in conventional stirred tank fermenters.
Although both C. cohnii and Schizochytrium are microalgae, they are heterotrophic
and therefore do not need light as a prerequisite for growth. As a result, both can be
grown in fermenters using similar technologies used for other microbial fermentations. One peculiarity of these organisms, however, is a consequence of their marine
heritage: Both organisms require the growth medium to contain a substantial concentration of salt (NaCl) in order to grow. Seawater contains 1819,000-ppm Cl,
which is not a problem in glass vessels, such as laboratory bench fermenters; however, it is a major problem at industrial scale where stainless steel vessels are the
norm. The standard grade of stainless steel, S 30400 (more normally referred to
as simply 304), which is relatively inexpensive and is used for a large percentage of
applications (approximately 50% of all stainless steel used is 304) from household
pans and cutlery to industrial plants. A total of 304 can withstand high Cl conditions for short periods of time, assuming thorough washing in between, but is susceptible to crevice corrosion at above 200-ppm Cl if contact is prolonged. The
higher grade of stainless steel 316 is also used for industrial plants, although less
often because of increased expense and can withstand Cl concentration up to 1000
ppm, still insufficient for cultivation of microorganisms in media with salt concentrations similar to seawater. As a result, specific media have been developed (66),
and strains of both organisms that are capable of growth in low salt conditions
have been identified.
As with the ARASCO process, the organism is cultivated in sequentially larger
vessels to ensure sufficient inocula size, to final production vessels up to 200 m3
(see Figure 2). The development of the culture is followed to ensure optimal conditions, and once sufficient biomass and oil have accumulated, the biomass is harvested by centrifugation, spray dried, and then hexane extracted using technology
essentially identical to that used for vegetable oils (50, 67). The resulting oil is a
PROSPECTS FOR PRODUCTION OF OTHER PUFAS BY MICROORGANISMS
145
transparent orange oil that is bland in taste and odor. Despite its high DHA content,
DHA-rich SCO and in particular DHASCOTM is very stable, far more stable than
fish oils (6769), to the extent that microbial oils have to be relatively badly abused
before they take on the taste/odor characteristics of even the most refined fish oils.
4. PROSPECTS FOR PRODUCTION OF OTHER PUFAS
BY MICROORGANISMS
4.1. Eicosapentaenoic Acid (20:5, n-3)
The markets for ARA and DHA are now well established in Europe and the United
Stated and are clearly being developed in Japan as well as in China. The next PUFA
that appears likely to be produced is EPA, which could be used as a nutraceutical
for over-the-counter sales or, more likely, as a possible pharmaceutical, as there
have been numerous reports of its benefits for the treatment of various diseases
and disorders. Such conditions as atherosclerosis, cancer, rheumatoid arthritis, psoriasis, bipolar disorder, schizophrenia, and Alzheimers disease have all been said to
have been improved or relieved by the oral administration of EPA, sometimes on its
own, although sometimes with DHA (34, 7072). EPA and DHA, of course, occur
together in many fish oils, and when treatment with both these PUFAs is advocated,
then the use of fish oils would seem to be the appropriate recommendation.
EPA has long been recognized as having anti-inflammatory properties and therefore has potential uses against autoimmune diseases such as arthritis. However,
inflammation is now being recognized as a major factor in the progression of heart
disease (73). Thus, the cardioprotective activity of EPA may be associated with its
anti-inflammatory properties. There is also some evidence that growth of some
mouse tumors can be decreased by oral administration of large doses of EPA
(7476). The EPA may work by affecting fatty acid uptake by the tumor cells
and, in particular, by inhibiting linoleic acid uptake, which is converted into the
13-hydroxy derivative that is a positive promoter of tumor growth (77).
Opportunities for producing EPA, besides purifying it from fish oils, which will
always leave some DHA behind, using micro-organisms are currently somewhat
limited. Mortierella alpina strain 1S-4, which is used commercially to produce arachidonic acid (see above) can produce EPA if it is grown during the lipid accumulation stages at 12 C rather than at the normal 28 C (78, 79). Also, EPA can be
produced in increased quantities if alpha-linolenic acid (18:3, n-3) is fed to this particular strain of M. alpina (80). Up to 42-mg EPA/g cells has been achieved. However, ARA is still present in all of these oils. Some species of the fungus, Pythium,
has also been shown to produce EPA at up to 34-mg/g cells (8183), but other
PUFAs, including arachidonic acid, are also synthesized simultaneously, making
production of an EPA-rich oil, devoid of ARA, somewhat difficult.
The best current sources of EPA would appear to be the photosynthetic microalgae, of which Porphyridium cruentum, Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum appear to be the prime candidates (8487).
All produce oils with EPA between 25% and 38% of the total fatty acids (see
146
Table 1), but in each case, there is always some ARA or DHA, or even both, that is
present. The oil content of these algae is also not very encouraging as it is generally
less than 20% of the cell mass. The oil is not high in triacylglycerols, which is the
desirable form for it, but it is a variety of complex lipids involved in the photosynthetic apparatus of the cells.
Thus, there is as yet no equivalent oil to the DHASCO from Crypthecodinium
cohnii, in which EPA is the sole PUFA that is present and moreover is present principally as its triacylglycerol. Some researchers, though, have advocated the use of
genetically engineered microorganisms to produce EPA by taking appropriate genes
from bacteria (a few of which can also produce EPA) and cloning them into more
amenable species, but this is far from an easy task if one also needs to increase the
total oil content of the species. There is considerable effort currently being placed
into finding suitable micro-organisms for the production of EPA-rich oils, and it is
not inconceivable that within the next 2 to 3 years, such organisms will be found,
but at the present time, no microbial source of EPA is in production.
4.2. Other PUFAs
Prospects of producing a variety of other PUFAs using microorganisms exist,
although whether any of these will reach commercial fruitition will depend on
the demand for such materials. In many cases, these unusual fatty acids can be produced using Mortierella alpina, which normally produces arachidonic acid (see
above). However, Shimizu et al. have shown (32, 88) that it is possible to mutate
this fungus so that the normal sequence of desaturation and elongation of the C18
PUFAs can be disrupted at various key points. Thus, mutants of this organism have
been created, which can accumulate:
Stearidonic acid (18:4, n-3) [here the elongation of gamma-linolenic acid
(18:3, n-6) to 20:3, n-6, had been prevented by a mutation that knocked out
the gene coding for this elongase enzyme activity; the GLA was now
desaturated with an existing delta-15 desaturase]. The production of this
PUFA has, however, also been demonstrated in transgenic crop plants by
Monsanto, so the potential for this PUFA as the basis of a SCO is limited.
Dihomolinolenic acid (DHGLA; 20:3, n-6) (where the final desaturation of
this fatty acid to arachidonic acid by a delta-5 desaturase had been either
deleted by mutation or blocked by the presence of a specific inhibitor of the
desaturase).
Eicosatrienoic acid (ETA; 20:3, n-9: Mead acid) (here a double mutant of the
parent fungus was produced that lacked both delta-12 and delta-15 desaturase
activities so that oleic acid, 18:1,n-9, could only be desaturated between its
existing double bond and the carboxylate head group).
Eicosatetraenoic acid (EteA; 20:4, n-3) [where the mutant that had accumulated DHGLA (see above) was now grown with linseed oil (as a source of
alpha-linolenic acid, 18:3, n-6) instead of the normal glucose; this fatty acid
was then channeled down the n-3 route of metabolism].
THE FUTURE OF MICROBIAL OILS
147
In all cases, the amounts of the various PUFA were relatively low, although for
DHGLA, the amount reached 23% of the total fatty acids. Some of the above
PUFAs also occur in small amounts in various marine microalgae, although no
routes to optimize their commercial productions have been indicated.
The unusual PUFA, docasopentaenoic acid (22:5, n-6), which occurs at about
12% of the total fatty acids of the Schizochytrium sp. used for DHA production (see
Table 4), is thought to be produced in Japan by Nagase-Suntory, possibly as a
byproduct from the purification of the DHA-rich oil that this organism produces,
although the exact means of production are not certain. The direct applications
of DPA are uncertain, although as discussed in Section 3.2.1, there may be some
benefits of including DPA along with both DHA and ARA as it may serve to maintain DHA at a higher circulating level (65).
5. THE FUTURE OF MICROBIAL OILS

The past two decades have seen the commercial productions of several single cell
oils: oils rich in gamma-linolenic acid, arachidonic acid, and docosahexaenoic acid.
Although gamma-linolenic acid rich-SCO was in production for only about 6 years
in the United Kingdom during the 1980s and is no longer considered an economic
reality, the production of DHA-and ARA-rich-SCOs has demonstrated the potential
of this technology, in dramatic style. These SCOs are enjoying a period of enormous growth in demand, to the point where the market is supply- rather than
demand-limited. The main global supplier of SCO, Martek Bioscience Corporation
(>95% of the global market in SCO are Martek-oils) is doubling its production
capacity every year for at least the next two years (2004 and 2005). As a consequence, Martek has become a major fermentation company with millions of Liters
of fermentation capacity solely dedicated to SCO production.
Against this background, it is more than likely that an SCO rich in eicosapentaenoic acid will also become commercially viable within the next few years as the
demands for improved supplies of this fatty acid are now beginning to accelerate.
Stearidonic acid could well be the next one after that, although in this particular
case, this PUFA can be produced using selected species of plants known as Echium,
and transgenic crop plants producing this fatty acid have been produced by Monsanto.
The microbial route to production will, however, always be expensive, and thus,
only the most expensive of the PUFAs will justify being produced by this means.
Fermentation technology is never going to be a cheap alternative to agriculture. The
logical progression, therefore, is to see the next phase of PUFA production moving
from micro-organisms toward the use of plants. This can, though, only take place
by genetic manipulation as no plants are known that produce ARA, EPA, or DHA
and it is these three PUFAs that are the major targets for production because of the
potential size (both in volume and in price) of the markets for each of them.
To design a plant that will produce these oils, it is necessary to clone into the
plant of choice (probably sunflower or rapeseedcanola) genes that will code
for one or two elongating enzymes (to convert C18 fatty acids into C20 fatty acids
148
and then to elongate the C20 fatty acids into C22 fatty acids) and a number of desaturases that will then convert a diunsaturated fatty acid (i.e., 18:2, which is the
major fatty acid of these plants) into, eventually, a hexa-unsaturated fatty acid. Thus,
to produce DHA (via a classic fatty acid route), a minimum of six different genes
will be needed and possibly more to ensure that these genes will function correctly.
The expression of the introduced genes must also be carefully controlled so that
the proteins (i.e., enzymes that they are coding for) will be specifically targeted
(both spatially and temporally) to ensure that the LC-PUFA are produced only in
seed tissue and only during the oil accumulation phase. Although this targeting is
not a major technical obstacle, the apparent innate inability of plants to metabolically process LC-PUFA could be a considerable challenge. Although the introduction of fatty acid desaturase genes (even those not usually fond in plants) leads to an
appreciable build-up in new unsaturated fatty acids, the introduction of an fatty acid
elongase has not yet engendered a significant accumulation of C20 or C22 PUFAs
(to the authors knowledge). It is possible that this failure to produce C20 PUFA is
not caused by a lack of elongase activity per se, but it is associated with the inability of plant acyltransferases to efficiently transport C20 fatty acids. The acyltransferases are enzymes involved in the shuttling of fatty acids into cellular
membranes for their final desaturation and then incorporation into triacylglycerols
(TAG). If this is the case, then several more genes (for LC-PUFA acyltransferases)
may need to be introduced into plants to facilitate LC-PUFA accumulation, and this
would add enormously to the complexity of producing a satisfactory GM plant for
LC-PUFA production.
Some of the problems associated with the transgenic production of LC-PUFA in
plants (although not the problems associated with fatty acid transport) may be
solved by harnessing the newly discovered PKS-like route for LC-PUFA biosynthesis. This route, completely separate from classic fatty acid biosynthesis,
is catalysed by a single enzyme complex, and not using fatty acid desaturases or
discrete elongases appears to operate in certain marine prokaryotes and in Schizochytrium (89). The PKS-like enzymatic machinery is encoded by three or four
open reading frames, decreasing the number of genes that would be required to be
transformed into a potential plant host. However, the very large size of these genes
could introduce problems of their own as it becomes increasingly more difficult to
clone genes as their size increases.
A further, and as yet unresolved, problem develops as to how the necessary reducing power that is needed in each of the elongating and desaturating reactions will
be generated. The plant of choice for genetic manipulation is already in metabolic
balance: It produces what it needs, no more, no less. When an increased metabolic
burden is then placed on a plant to produce products that require additional
resources from the central metabolic pathways, it is not clear how these resources
will be achieved. An increased demand for reducing power in one part of the plant
means that metabolic economies will have to take place elsewhere. Plants do not
have the capacity to increase their metabolic capacity at the whim of genetic
engineers. They are governed by the availability of light and of CO2. Thus, how
the plants that are being designed to produce high contents of PUFAs, particularly
ACKNOWLEDGMENTS
149
DHA, will be able to achieve this is, at least to the present authors, far from clear. It
is not beyond the bounds of possibility that such genetically modified plants may
produce much less oil than the normal plant as the energy and reducing power
needed (by the desaturase and elongase reactions) to produce the PUFAs must
come from the same sources that are being used to synthesize the normal fatty
acids. The situation in plants may parallel what was found with the production of
GLA in Mucor spp. (see Section 2.2.1): You can either find strains that produce a lot
of oil but have little GLA, or you can find strains that produce a lot of GLA but have
little oil, but you cannot have both occurring simultaneously. It may then take additional genetic manipulations to correct this imbalance, but distortion of one metabolic pathway can only be achieved at the expense of another so this is not likely to
be a trivial task.
Thus, although the GM plant route to PUFA formation is attractive, it is by no
means going to be as simple a task as the molecular biologists would appear to consider. One can expect that it will take the labors of many people many years to
achieve these objectives, but, given the high rewards that are at stake, it may
seem to many people just a question of time before the genetic manipulators are
successful. The question then that microbial oil producers have to decide is just
how long they have before the GM plant people achieve their goals, for when
that happens, it will be the end of SCO productions. They will simply be too expensive to compete. The pessimists would suggest no more than 10 years; the optimists
might suggest 20 or even 30 years.
But there is one final question that no one can yet resolve. Will GM plants be
accepted for the production of PUFAs? The public opinion of GM crops is, at
present, very much against their use in Europe. A survey conducted in the
United Kingdom during 2003 has indicated that over 80% of the population is
opposed to the introdution and use of GM crops, with only about 7% of the population being willing to consume any such GM product (90). The view against the use
of GM plants, which has no scientific basis but is wholly based on irrational fears,
now appears to be spreading to the United States and the rest of the world. If there
should be a moratorium against the use of GM crops in general, then it may be
that the industrial companies who are currently funding much of this work will
pull out these endeavors. If this should happen, no matter how regrettable this might
be scientifically, then this would spell the end of GM plant PUFAs. If this should
happen, the only way in which the demand for these desirable oils is going to be
met will be by the microbial route. Single cell oils could then have a long and distinguished future ahead of them.
ACKNOWLEDGMENTS
We are extremely grateful to Professor Sakayu Shimizu, Kyoto University, Japan,
for providing information about the current commercialization of microbial oils in
Japan. We are also indebted to Dr. David Kyle, Advanced BioNutrition Corp., MD,
for his perceptive reading of this manuscript and for his shrewd comments.
150
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5

Oils from Microorganisms

Martek Biosciences Corporation

1. GENERAL INTRODUCTION AND BACKGROUND

OILS FROM MICROORGANISMS

1.2. Microbial Oils

GENERAL INTRODUCTION AND BACKGROUND INFORMATION

biosynthesis is then used, in some cases, for the overproduction of triacylglycerols

OILS FROM MICROORGANISMS

GENERAL INTRODUCTION AND BACKGROUND INFORMATION

Figure 2. A diagrammatic presentation of a fermentation system for the production of a single

C. Microalgae and thraustochytrids3

OILS FROM MICROORGANISMS

are provided in other reviews (46) and in an earlier comprehensive monograph on

Figure 3. Stylistic presentation of the course of lipid accumulation by an oleaginous

GENERAL INTRODUCTION AND BACKGROUND INFORMATION

OILS FROM MICROORGANISMS

to give them increased quantities of lipid and, simultaneously, to produce more of

2. COMMERCIAL MICROBIAL OILS

2.1.1. Fungal Oils Rich in Gamma-Linolenic Acid

TABLE 2. Fatty Acid Profiles of GLA-SCOsMicrobial Oils Rich in Gamma-Linolenic

Production organism used by J & E Sturge Ltd., Selby, N. Yorks., UK.

18:3 18:3 20:1 22:1

COMMERCIAL MICROBIAL OILS

OILS FROM MICROORGANISMS

COMMERCIAL MICROBIAL OILS

OILS FROM MICROORGANISMS

COMMERCIAL MICROBIAL OILS

OILS FROM MICROORGANISMS

SCOS IN CURRENT (2003) PRODUCTION

3. SCOS IN CURRENT (2003) PRODUCTION

OILS FROM MICROORGANISMS

SCOS IN CURRENT (2003) PRODUCTION

OILS FROM MICROORGANISMS

SCOS IN CURRENT (2003) PRODUCTION

Production organism: Mortierella alpina (see 55).

SCOS IN CURRENT (2003) PRODUCTION

OILS FROM MICROORGANISMS

PROSPECTS FOR PRODUCTION OF OTHER PUFAS BY MICROORGANISMS

OILS FROM MICROORGANISMS

THE FUTURE OF MICROBIAL OILS

5. THE FUTURE OF MICROBIAL OILS

OILS FROM MICROORGANISMS

OILS FROM MICROORGANISMS

24. M. Beavan, A. Kligerman, R. Droniuk, C. Drouin, B. Goldenberg, A. Effio, P. Yu,

OILS FROM MICROORGANISMS

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