BioMatNet Item: FAIR-CT96-3146 - Production of polyunsaturated fatty acids (PUFAs) by algae: a complete bioprocessing concept for the large-scale production of high quality DHA containing oils

FAIR-CT96-3146
Production of polyunsaturated fatty acids (PUFAs) by algae: a complete bioprocessing concept for the large-scale production of high quality DHA containing oils

Contacts

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Biological Conversion : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Fine Chemicals : Pharmaceuticals/Cosmetics : Vegetable Oil/Fat

	Type of Project	Shared Cost
	Contract No	FAIR-CT96-3146
	Total Cost	1 382 500 ECU
	EC Contribution	849 500 ECU
	Start Date	01/09/1997
	Duration	36 Months

Objectives
The main objective of the current proposal is to develop a complete, reliable and reproducible large scale fermentation and bioprocessing concept for the microbial production of polyunsaturated fatty acids (PUFAs), yielding high quality docosahexaenoic acid (DHA)-containing oil that can be used for (Infant) food or pharmaceutical applications. DHA is the most abundant PUFA in the grey matter of the brain and also in the outer rod segments of the retina of the eye. Therefore, DHA is thought to be essential for proper brain and vision development of infants. Although cold water marine fish and egg yolk are known sources of DHA, these sources are not so suitable for human consumption. Therefore, other sources are now actively being sought for commercial, large scale production of DHA containing oils.

World-wide there is an increasing scientific and commercial interest for development of products and technologies with new micro-organisms. Within this field, our research focuses on economic PUFA production with algae. Crypthecodinium cohnii, a heterotrophic alga which is considered the most potentially useful DHA producing microorganism, will initially be cultivated at laboratory scale. Optimal conditions for growth and oil quality will be established by application of different culture media and different fermenter types. In addition, conversion of novel substrates/precursors like e.g. lineseed oil into DHA will be studied.

Semi-industrial scale fermentation and bioprocessing of the DHA-containing oil will be achieved by a stepwise approach. Optimal fermentation, harvest and oil-processing conditions, as established at laboratory scale, will be used as input for fermentation and down stream processing at subsequent 150, 1,500, and finally at 30,000 l scale. At these different scales, the effect of agitation conditions and the use of piched blade or marine impellers, or Rushton-type high efficiency turbines on biomass yield and oil quantity and oil quality will be studied. Oils will be extracted with traditional hexane extraction methods, but also new solvents like isopropanol will be included, as well as supercritical C0₂ extraction. The ultimate goal of the project is to provide, at the end of the research, a Standard Operating Procedure (SOP) for production, recovery and formulation of DHA. With this procedure trial quantities of oil in batches of 100 to 500 kg per run should be produced and costs/benefits diagrams established.

PUFA's in general, and DHA in particular have large commercial potential for application in e.g. infant-food, with an expected mark-et of at least 100 to 500 tonnes per year, due to their important role in infant visual and neural development.

Introduction
Long-chain polyunsaturated fatty acids of the omega 3 and omega 6 series are straight chain carboxylic acids of 20 or more carbon atoms that contain 3 or more double bonds. LC-PUFAs are important for human health as they are structural components of the cell membrane and the precursors of thromboxanes, prostaglandins and leukotrienes. At present, arachidonic acid (ARA, C20:4 omega 6), eicosapentaenoic acid (EPA, C20:5 omega 3) and docosahexaenoic acid (DHA, C22:6 omega 33) are considered the most important LC-PUFAs. LC-PUFAs can de novo be synthesised from the parent essential fatty acids linoleic (18:2 omega 6) and alpha-linolenic (18:3 omega 33) acids by desaturation and chain elongation but the overall capacity of humans to synthesize e.g. DHA is relatively low. Therefore, most of the DHA in our bodies is obtained from dietary sources, or, for a foetus or breast fed baby, from the mother. Recently, well documented research has indicated the importance of DHA in normal neurological development of infants. Based on these data the FAO/WHO has recommended the inclusion of supplemental DHA (and ARA) in both preterm and full term infant formulas. Although oils derived from fatty fish like herring, mackerel, sardine or salmon contain DHA and EPA these oils are often unsuitable for human consumption or for inclusion in infant formula. Therefore other sources, e.g. microbial PUFA producers, are now actively being sought for commercial large scale production of DHA-containing oils.Worldwide there is an increasing scientific and commercial interest for development of products and technologies with new micro-organisms. Within this field, our research focuses on economic PUFA production with algae.

Materials and methods
Crypthecodinium cohnii, a heterotrophic alga which is considered the most potentially useful DHA producing micro-organism, will initially be cultivated at laboratory scale. Optimal conditions for growth and oil quality will be established by application of different culture media and different fermenter types. In addition, conversion of novel substrates/precursors into DHA will be studied. Semi-industrial scale fermentation and bioprocessing of the DHA-containing, oil will be achieved by a stepwise approach. Optimal fermentation, harvest and oil-processing conditions, as established at laboratory scale, will be used as input for fermentation and down stream processing at subsequent 150, 1,500, and finally at 30,0001 scale. At these different scales, the effect of agitation conditions and the use of different mixing strategies on biomass yield and oil quantity and oil quality will be studied. Oils will be extracted with traditional extraction methods, but also new solvents like isopropanol will be included, as well as supercritical CO₂ extraction.

Results
The ultimate goal of the project is to provide, at the end of the research, a Standard Operating Procedure (SOP) for production, recovery and formulation of DHA. With this procedure trial quantities of high quality docosahexaenoic acid (DHA)-containing oil that can be used for (infant) food or pharmaceutical applications in batches of 100 to 500 kg per run should be produced, and costs/benefits diagrams established. Preliminary cost calculations indicate that the final biomass, total fermentation time and the lipid content of the organism are very important for the economic feasibility, as these factors largely determine the productivity. Environmental parameters like the amount of sugars and nitrogen appear to have a significant effect on growth and lipid accumulation of a heterotrophic marine alga C. cohnii.

Dissemination activities
The following paper has been published:

Sijtsma, L., de Swaaf, M. & Eggink, G (1988). Microbial production of long chain polvunsaturated fatty acids. 7th Netherlands Biotechnology Congress, Ede, The Netherlands

Contacts

Coordinator

Lolke SIJTSMA / Wageningen University and Research Centre Agrotechnological Research Institute (ATO BV) / NETHERLANDS

Participant