Eskimos had a favourable lipid profile with low levels of triglycerides, plasma cholesterol and very low-density lipoproteins (VLDL) and high levels of high-density lipoproteins (HDL) (Dyerberg et al. 1975). As the Eskimos consume a large amount of marine mammals and arctic fish in their diet, which are rich in n-3 fatty acids, these PUFA were presumed to play a major role in the health effects of fish oil. Since then several epidemiological and medical studies have been performed to investigate the beneficial effects of n-3 PUFA on humans (Kromhout et al. 1985; Daviglus et al. 1997; Albert et al. 1998).
DHA, for instance, attracted much attention because of its various physiological functions in the human body. DHA reduces or inhibits risk factors involved in various diseases such as cardiovascular diseases (Kromann and Green 1980; Kang and Leaf 1996; Nordùy et al. 2001) and has some positive effects on diseases such as hypertension, arthritis, arteriosclerosis and thrombosis (Horrocks and Yeo 1999). Furthermore, DHA is an essential component of cell membranes in some human tissues and, for instance, accounts for over 60 per cent of the total fatty acids in the rod outer segment in the retina (Giusto et al. 2000). DHA is regarded as essential for the proper visual and neurological development of infants because of its roles as structural lipid component (Nettleton 1993; Crawford et al 1997; Das and Fams 2003). As pre-term and young infants are unable to synthesise DHA at a fast enough rate to keep up with the demand from the rapidly growing brain (Crawford 1987) they must obtain these compounds from their diet. In general, breastfeeding serves as a good source of PUFA (Huisman et al. 1996). However, although it has been recommended that all infant formulas include DHA (FAO/WHA Expert Committee 1994), application of DHA in some infant formulas only started recently. EPA is the precursor of a family of eicosanoids that are widely involved in metabolic regulation (Hwang 2000). Some studies also suggest that EPA is a potential anticachexia and anti-inflammatory agent (Calder 1997; Gill and Valivety 1997; Babcock et al. 2000).
With respect to the biological function of PUFA the position of the double bond strongly affects the properties of the fatty acids. For instance, eicosanoids derived from the n-6 polyunsaturated fatty acid arachidonic acid (AA, 20:5 05,8,11,14) have strong inflamatory properties, whereas those produced from EPA are anti-inflammatory (Gill and Valivety 1997).
Although the optimal intake of PUFA has not yet been established, there is some consensus that the PUFA intake should be at least 3 per cent of the total lipid intake (Gill and Valivety 1997). Studies suggest that while total fat levels in the typical Western diet are too high, the intake of long-chain n-3 PUFA is too low (Newton 1998). At present, most consumed PUFA originate from plant oils and belong to the n-6 group. The excess of n-6 fat intake compared with n-3 intake has practical consequences because, as they are very similar except for the position of one double bond, they may compete for the same enzymes that metabolise them. An excess of n-6 over n-3 fatty acids leads to poor metabolism of ingested n-3 fatty acids to the longer n-3 fatty acids EPA and DHA (James et al. 2000). In order to improve the balance generally seen as optimal for human health, an increase in n-3 PUFA consumption and a reduction in n-6 PUFA is needed. The British Nutrition Foundation recommended a n-6 to n-3 PUFA ratio between 5:1 and 3:1 (British Nutrition Foundation 1992).
Although plant materials such as flaxseed, canola and soybean oil contain the n-3 PUFA a-linolenic acid, this paragraph will focus on the n-3 fatty acids with 20 and 22 carbon atoms. Currently, the main sources of DHA and EPA are fatty fish species such as herring, mackerel, sardine and salmon (Gunstone 1996), as their flesh usually contains a high proportion of fat tissues. The quality of the fish oil, however, is variable and depends on fish species, seasons and location of catching sites. The application of fish oil PUFA in foods, for inclusion in infant formulas, or for pharmaceutical applications may have some disadvantages because of contamination of the fish oil by environmental pollution such as PCBsm(polychlorinated biphenyls) or dioxin-like compounds and problems associated with the typical fishy smell and unpleasant taste. Furthermore, as marine fish oil is a complex mixture of fatty acids with varying lengths and degrees of unsaturation, expensive purification may be required before application.
At present the fish oil production amounts to about 1.1 million tonnes annually (Gunstone 2001), of which 70 per cent is utilised for production of fish feed for farmed fish (Tuominen and Esmark 2003). The demands for n-3 PUFA are rapidly increasing owing to a rapid increase in aquaculture and application in food and pharmacy. It is therefore expected that within 10 years the production of PUFA from current sources will become inadequate for supplying the expanding market. In order to meet the expected rise in demand and to circumvent the detrimental aspects of fish oils, alternative production processes for PUFA are currently being developed. These include the development of refining techniques of fish oils (Yamamura and Shimomura 1997) and the exploitation of microbial PUFA sources (Barclay et al. 1994; Kyle 1996; Ratledge 2001; de Swaaf 2003) which may offer a sustainable production of n-3 PUFA.
16.2.2 Microbial production of PUFA
Although marine fish and mammals appear to have some capacity for de novo biosynthesis of n-3 PUFA, the majority of the PUFA in their body originates from their diet. Fish consume marine zooplankton that have fed on phytoplankton (Ackman et al. 1964) such as bacteria, lower fungi, microalgae and some microalgae-like organisms. These organisms are known as the primary producers in the marine food chain and they are the actual primary synthesisers of PUFA (Yap and Chen 2001).
In human and animal nutrition, lipids have been obtained traditionally from plant and animal sources. However, some valuable lipids are now being produced from micro-organisms. As a source of oil or, in more general terms, lipids, micro-organisms are less well known than plants and animals. Microbial oil or single cell oil (SCO) production is a relatively new concept, first proposed in the twentieth century (Ratledge 2001). Microbial oils may be produced in stirred bioreactors in the dark with an organic carbon source and sufficient amounts of minerals, nitrogen, oxygen and micronutrients by so-called heterotrophic micro-organisms. Alternatively phototrophic species may be cultivated under light in open or closed systems but this process is less well established for SCOs. Upon harvest lipids may be extracted from the dried biomass, formulated and used for their different applications.
As the prices for most bulk plant oils are relatively low, and animal fats are even cheaper, it is likely that processes for the microbial production of oils should focus on high value added products. Although technically feasible, earlier attempts to commercially produce SCOs have failed because of economics (Davies 1992; Nakahara et al. 1992; du Preez et al. 1995; Ratledge 2001). However, the SCO concept has now yielded several successes with regard to PUFA and industrial interest is increasing (Barclay 1991; Barclay et al. 1994; Kyle 1994, 1996, 1997; Ratledge et al. 2001b; de Swaaf 2003).
Based on their percentage of n-3 PUFA, oleaginous marine micro-organisms such as microalgae or marine fungi may be interesting alternatives for fish oils. At present the contribution of microbial PUFA to the oil industry is nearly negligible but there are several reasons to increase their use in the near future. In heterotrophic systems microbial oils can be produced all over the year as these processes are usually independent of light, and temperature can be well controlled. Another advantage is that microbial oils are free from contaminants such as PCBs and dioxin-like compounds. Compared with fish oil, microbial oils often contain high levels of the desired fatty acids and, because of their lipid composition, purification of PUFA from microbial oils may be easier or not required.
Micro-organisms capable of producing n-3 PUFA above C20 include lower fungi, bacteria and marine microalgae (Bajpai et al. 1991; Kendrick and Ratledge 1992; Gunstone et al. 1994; Kyle 1996, 1997; Vazhappily and Chen 1998; Ratledge 2001; de Swaaf 2003). Bacteria, however, are probably not suitable as PUFA producers, as they do not accumulate high amounts of triacylglycerols and may contain unusual fatty acids and lipids not found in other systems (Ratledge 2001).
Oleaginous micro-organisms could provide an economically feasible source of PUFA, provided that most of the PUFA occur in triacylglycerols which is the preferred form to take lipids in the diet (Kendrick and Ratledge 1992). Furthermore, micro-organisms preferably contain one specific PUFA rather than a mixture of various acids. This gives the microbial oils an additional value as compared to fish oils, which contain mixtures of PUFA. The development of a microbial PUFA production process requires the selection of the proper microorganism and optimised cultivation techniques (Ratwan 1991). As both, EPA and DHA are important nutritional n-3 PUFAs much effort has been devoted to finding a commercial source of these fatty acids other than from fish oil.
At present a few photoautotrophic systems are being used for cultivation of microalgae. The oldest and simplest systems for cultivation of phototrophic algae are open ponds. These cultivation systems, however, are dependent on the weather and climate and therefore the product quantity and quality of separate batches is variable. Processes are time consuming owing to the low specific growth rates of algae, and available light limits the attainable biomass concentrations. Because of contamination with bacteria and predation by protozoa, phototrophic cultivation in open ponds is feasible only when suitable selective environments can be used (e.g. high salinity, high pH). In addition, optimal culture conditions are difficult to maintain and, because of the low biomass concentrations, harvesting costs are relatively high (Barclay et al. 1994; Molina Grima et al. 2003). In closed photobioreactors, made of transparent materials and generally placed outdoors for illumination with sunlight, the environmental parameters can be better controlled, allowing for higher biomass concentrations and a reduced contamination risk. Scale-up of the process is, however, limited by the ability to effectively introduce the light (Pulz 2001) and, in general, the costs of alga production in mass culture in such fermentors are high (Molina Grima et al. 2003).
Percentages of specific fatty acids in the lipids of selected marine micro-organisms* It was not described whether the form was n-3 or n-6.
a Singh and Ward (1996); b Yokochi et al. (1998); c de Swaaf et al. (1999); d Vazhappilly and Chen (1998); e Molina Grima et al. (1993); f Servel et al. (1994); g Viso and Marty (1993); h Meireles et al. (2002); i Cohen (1999); J Wen and Chen (2000).
H and P indicate heterotrophic and phototrophic growth, respectively.
For commercial PUFA production, heterotrophic production systems, where microalgae are growing on reduced carbon sources, have been considered for production of specialty SCO (Barclay 1991; Kyle 1994, 1996; Mukherjee 1999). In heterotrophic cultures (i) optimal and axenic conditions can be maintained (Chen 1996), (ii) oil production can be carried out throughout the year as there is no seasonal or climatic dependence, (iii) the process can be controlled and product quality guarantees can be given, (iv) high cell densities, over 100 g dry weight/L, can be achieved (de Swaaf et al. 2003a) and (v) technology able to deal with heterotrophic fermentation is widely available.
For n-3 PUFA production by heterotrophic marine micro-organisms, however, several challenges must also be faced:
At present, only a limited number of heterotrophic species that accumulate n3 PUFA are available.Due to the required rich media and the relatively low growth rates of marine micro-organisms the risk of contamination is an issue.Economics of production should be in good proportion to market prices.Furthermore, for all new products from microalgae legislation and safety items need to be considered.
Most of the EPA production processes studied to date have been based on photoautotrophic growth (Qiang et al., 1997; SaÂnches MiroÂn et al. 2002; Molina Grima et al. 2003,. Unfortunately, the EPA yield and productivity in photosynthetic systems are low. In a closed flat plate reactor with a narrow light-path and intensive stirring which facilitated high cell concentration, a maximal EPA productivity of 58.9 mg/L/day, corresponding with 2.4 mg/L/h, was produced in Monodus subterraneus (Qiang et al., 1997). These values, however, are probably far too low in order to establish processes for economically feasible EPA production by photosynthetic microalgae.
Recent advances in heterotrophic production of EPA, with an emphasis on the use of diatoms as producing organisms, were recently reviewed by Wen and Chen (2003). By using glucose as carbon source and nitrate as nitrogen source for the diatom Nitzschia laevis, an optimal EPA yield of 695 mg/L in 14 days of a fed-batch cultivation was reported (Wen et al. 2002). Although, compared with batch cultivation the use of a fed-batch cultivation remarkably improved EPA productivity (2.1 mg/L/h), these values are comparable with those reported for Monodus under photoautotrophic growth and still rather low for commercial production.
The exploitation of marine micro-organisms for the production of DHA will be discussed in the next section in more detail.
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