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5
Oils from Microorganisms

James P. Wynn1 and Colin Ratledge2
Martek Biosciences Corporation Columbia, Maryland 2 Lipid Research Centre, University of Hull, Hull, United Kingdom
1
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 eld 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 dened 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.
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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 xed 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 rst 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 rst 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 modied by genetic manipulation where genes (that is, specic 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
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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 gure 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,
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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 rst 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 eld 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 eld 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 rst decade of the twentyrst 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 specic 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, sh. Fish, though, are a dwindling resource and the oils, which are mainly obtained from their livers or the bodies of fatty sh, 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
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Acid cryovial
Base Antifoam
CENTRIFUGE Waste
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 nally 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 sunowers 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 denition. 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 prole of the constituent fatty acids. More detailed lists
TABLE 1. Lipid Contents and Fatty Acid Proles 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) Lipid Content (% w/w) 42 65 58 64 63 66 72 45 36 14:0 16:0 16:1 (n-7) 44 12 32 37 34 18 37 12 11 5 1 4 6 3 1 6 18:0 18:1 (n-9) 31 73 44 48 51 66 47 50 28 18:2 18:3 (n-6) (n-3) 9 12 8 3 3 8 12 51 1 1 18:3 (n-6) 20:4 (n-6) 20:5 (n-3) 22:6 (n-3)
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 Entomophthora coronata Cunninghamella japonica Mortierella isabellina Rhizopus arrhizus Mucor alpine-peyron Ascomycetes Aspergillus terreus Fusarium oxysporum Pellicularia practicolo
Others (%) 23:0 (3%) 24:0 (6%)
Trace Trace Trace Trace Trace Trace Trace Trace Trace
8 3 15 7 5 3 3 22 1
26 43 60 86 57 38
1 31 Trace 1 19 10
23 9 16 29 18 15
15 2 14 3 6 7
25 14 48 55 22 30
1 2 14 3 10 9
4 1 8 3 12 1
4 5
Notes :
20:1 (13%) 22:1 (8%) 12:0 (40%) 20:0 (8%) 20:3 (6%)
57 34 39
2 Trace Trace
23 17 8
Trace 14 8 20 2 11
40 46 72
21 5 2
Hyphomycetes Cladosporium herbarum Clavicipitaceae Claviceps purpurea
49 60
Trace Trace
31 23
12 2
35 19
18 8
12-HO18:1 (42%) Continued
C. Microalgae and thraustochytrids3 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 28 52 50 23 20 45 24 5 40 25
8 1 12 16 12 4 17 3
63 26 13 16 16 10 19 15 10 30 32 8
2 5 21 2 1 11 10 22 21 5 8
4 23 1 58 21 3 5 3 1 <1 5 16
9 10 2 9 1 2 2 1 4 5 2
14
12 21 <1 1 1 2
3 <1 14 4 1 16 3
45 25 34 38 33 38 1
40 11 4 28 52
18:4 (11%) 14:1 (13%) 22.5 (n-5) (8%)
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.
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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 rst 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.
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Under the conditions of nitrogen-limited growth, the rst 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 . . . . . . . . . . . . 2
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 nal 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 identication 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
2.1.1. Fungal Oils Rich in Gamma-Linolenic Acid
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 identied. 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 efcacy 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 efcacy 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
TABLE 2. Fatty Acid Proles 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 2
16:1 1 1 1
18:0 6 6 5 10 2 4 1
18:1 40 44 51 32 8 16 10
18:2 (n-6) 11 12 10 12 75 40 48
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
25 ND $40 30 16 30 30
22 27 24 25 6 10 6
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%.
COMMERCIAL MICROBIAL OILS
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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 identied 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 satised: 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 difcult, and the costs of oil production would be increased. Equally, the extracted oil should be at least 90% as a triacylglycerol so that subsequent renement 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, puried 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 nally chosen production organism, Mucor circinelloides, satised 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 prole 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 rst 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 conguration 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 insufcient 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 ercer 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 specic 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 nancially subsidized from the Common Agricultural Policy. This meant that growers of evening primrose oil received cash benets 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 nancially 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
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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 articially high price of the fermentation feedstock. The nal blow to the production of the fungal oil came with the introduction of borage oil (Borago ofcinalis) 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 difcult 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 nancial benets that had accrued from the EU Common Agriculture Policy for evening primrose cultivation. In 1990, and against this background of increasing erosion of the prot 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 difculties in extracting the oil (a process using hexane was used), and the oil could be easily puried 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 xed 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 sufcient margin of prot was considered possible if a microbial oil could be produced that mimicked the fatty acid prole of cocoa butter and, most importantly, had the same melting prole as cocoa butter. This manifests itself
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TABLE 3. Fatty Acid Proles of CBE-SCOsMicrobial Oils for use as a Cocoa Butter Equivalentin Comparison with Cocoa Butter. Relative % (w/w) of Major Fatty Acids 16:0 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
WT wild type.
a
18:0 15 24 47 25 31 13 38 25 32-37
18:1 45 48 25 33 30 47 16 38 30-37
18:2 5 3 8 7 6
18:3 (n-3) 0.5 1 2 1 7 14 1
24:0 2 2 4 1
Ref 27 28 21, 23 21, 23 22, 23 24 24 29
30 18 15 33 24 30 26 26 23-30
6 2-4
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
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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 prole (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 nd their way into the nal 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 prole 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 nal 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, specically, genetically modied 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
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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 sufciently to cause the desired effects. The fatty acid prole 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 rened 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 prot, 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 sufciently 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
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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-dened and publicly recognized benets 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 benecial physiological effects of ARA and DHA. Animals (including sh) 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 proles, 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 sh, 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 sh livers have added another dimension to the argument against the use of sh oils. Fish livers, together with whole sh 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
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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 rst 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 exemplied 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.
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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 gure 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 denite historical association to any food product, extensive toxicological studies were performed to conrm 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.
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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 eld. 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 difculties 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 certied stock culture of the production strain of Mort. alpina. The thawed cryovial is used to inoculate a series of shakeask 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 laments or as pellets (of varying sizes; depending on conditions). Although feather-like hyphal laments 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
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viscosity of the culture to increase and agitation of the culture to be difcult (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 lter pressing, and dried before the oil can be extracted and puried using techniques essentially identical to those used for vegetable oil production. The resulting oil is a pale yellow brilliant oil with a relatively bland avor that is remarkably resistant to oxidation. The fatty acid prole of the commercial oil is given in Table 4 (55). 3.2. Docosahexaenoic Acid The most unsaturated fatty acid found in signicant 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 deciency 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 sh oils; however, the use of sh oil as a DHA supplement is problematic. Fish, like all animals, possess fatty acid proles that, to a large extent, reect their dietary intake rather than metabolic ability to synthesize fatty acids. As a result, the fatty acid proles of sh 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 sh 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 benecial effects of ARA-rich SCO supplementation of infant formula and is associated with growth retardation in neonates (57).
TABLE 4. Fatty Acid Proles of Microbial Oils Rich in Arachidonic Acid and Docosahexaenoic Acid that are Produced Commercially (from 55). Fatty Acid Composition (Rel. % w/w) 12:0 Oil ARASCOa DHASCOb Schizo-SCOc
a b c
14:0 0.4 20 13
16:0 8 18 29
16:1 0 2 12
18:0 11 0.4 1
18:1 14 15 1
18:2 (n-6) 7 0.6 2
18:3 (n-6) 4
18:3 (n-3) 3
20:3 (n-6) 4 1
20:4 (n-6) 49
22:5 (n-6) 12
22:6 (n-3) 0 39 25
24:0 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.
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Other problems associated with the administration of sh oil to the general population, and in particular to neonates, is the potential for the presence of environmental toxins in sh oils. As the sh 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 sufcient amounts to cause concern that the British Government commissioned a report that has highlighted the extent of contamination of sh stocks (58). In particular, the presence in sh oils of dioxins and polychlorinated biphenyls (PCBs) was noted and the report recommended that oily sh should not be eaten more than once a week. More recently, the specter of mercury in sh 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 sh intake. Furthermore, a recent study has determined that the mercury content of sh oil diminishes the cardiovascular benets of these oils (60). Any inclusion of the oils from such sh, 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 sh 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 signicant 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 sh oil (62). Two microalgal sources, in particular one dinoagellate (Crypthecodinium cohnii) and one chytrid (Schizochytrium), have been found to synthesize sufcient 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 proles of the resultant oils that have signicant 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 signicant 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 sufcient 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 benets of including DPA in a dietary supplement of ARA DHA, and this would occur by using the Schizochytrium oil along with the ARA-SCO.
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Initially, the two organisms were developed as competing processes by two pioneering U.S. rms, 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 insufcient for cultivation of microorganisms in media with salt concentrations similar to seawater. As a result, specic media have been developed (66), and strains of both organisms that are capable of growth in low salt conditions have been identied. As with the ARASCO process, the organism is cultivated in sequentially larger vessels to ensure sufcient inocula size, to nal production vessels up to 200 m3 (see Figure 2). The development of the culture is followed to ensure optimal conditions, and once sufcient 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
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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 sh 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 rened sh 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 benets 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 sh oils, and when treatment with both these PUFAs is advocated, then the use of sh oils would seem to be the appropriate recommendation. EPA has long been recognized as having anti-inammatory properties and therefore has potential uses against autoimmune diseases such as arthritis. However, inammation 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-inammatory 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 sh 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 difcult. 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 nding 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 nal 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 specic 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 purication 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 benets 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 sunower 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 specically 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 signicant 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 efciently transport C20 fatty acids. The acyltransferases are enzymes involved in the shuttling of fatty acids into cellular membranes for their nal 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 difcult 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 modied 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 nd strains that produce a lot of oil but have little GLA, or you can nd 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 nal 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 scientic 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 scientically, 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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Oils from Microorganisms

OILS FROM MICROORGANISMS

GENERAL INTRODUCTION AND BACKGROUND INFORMATION

OILS FROM MICROORGANISMS

GENERAL INTRODUCTION AND BACKGROUND INFORMATION

125 ml To All Fermentors SPRAY DRYER 1L

200 L 2,000 L 12,000 L 120,000 L

PACKAGING DRY BIOMASS

Others (%) 23:0 (3%) 24:0 (6%)

Trace Trace Trace Trace Trace Trace Trace Trace Trace

Hyphomycetes Cladosporium herbarum Clavicipitaceae Claviceps purpurea

12-HO18:1 (42%) Continued

18:4 (11%) 14:1 (13%) 22.5 (n-5) (8%)

OILS FROM MICROORGANISMS

GENERAL INTRODUCTION AND BACKGROUND INFORMATION

OILS FROM MICROORGANISMS

COMMERCIAL MICROBIAL OILS

OILS FROM MICROORGANISMS

COMMERCIAL MICROBIAL OILS

OILS FROM MICROORGANISMS

18:3 (n-3) 0.5 1 2 1 7 14 1

Ref 27 28 21, 23 21, 23 22, 23 24 24 29

COMMERCIAL MICROBIAL OILS

OILS FROM MICROORGANISMS

SCOS IN CURRENT (2003) PRODUCTION

OILS FROM MICROORGANISMS

SCOS IN CURRENT (2003) PRODUCTION

OILS FROM MICROORGANISMS

SCOS IN CURRENT (2003) PRODUCTION

18:2 (n-6) 7 0.6 2

SCOS IN CURRENT (2003) PRODUCTION

OILS FROM MICROORGANISMS

PROSPECTS FOR PRODUCTION OF OTHER PUFAS BY MICROORGANISMS

OILS FROM MICROORGANISMS

THE FUTURE OF MICROBIAL OILS

OILS FROM MICROORGANISMS

OILS FROM MICROORGANISMS

OILS FROM MICROORGANISMS

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