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Heterotrophic Polyunsaturated Fatty Acids - project proposal 15-11-2010

Emile Anton Esterhuizen Introduction Polyunsaturated fatty acids, in particular the essential omega-3 fatty acids, eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), commonly known as omega oils, are essential in the human diet. They serve many physiological functions in the human body including, brain and retinal development in infants, proper brain functioning in adults, and even in the prevention of heart disease(8). The main dietary source of these fatty acids is fish oils, particularly high concentrations of these fatty acids are found in salmon and cod liver oil, examples of which are products such as “Möllers Tran.” Although many fish species contain high concentrations of omega oils they are not able to synthesise these oils and so require a dietary source. In aquaculture, omega oils are provided through a number of sources; microalgae in the early hatchery stages and then later from by-catch and waste fish resources in the form of fish oil(2). There is a crucial sustainability issue in modern fisheries and aquaculture. The world is becoming ever more reliant on aquaculture to meet its dietary demands due to the dwindling global supplies of wild-caught fish. Alarmingly aquaculture is becoming more reliant on wild-caught fish (in the form of waste fish and by-catch) to meet its dietary supplies(1), where wildcaught fish oils are required in the formulation of fish feed. New bulk production technologies are required to ensure that an economically viable method of production can be achieved for the production of omega oils and already substantial progress in this field has been made. The primary producers of these oils are microalgae(3)(4)(5). There are many species that contain these omega oils, Isochrysis galbana, Phaeodactylum tricornutum, Schizochytrium SR21, Crypthetocodinium cohnii(3)(5), to name a few. However only a few species are known to overproduce omega oils and then it is often very specific omega oils, for example C. cohnii, is able to produce a cell biomass (over 150g/L)(8) containing an extremely high lipid content, with the majority of the fatty acids present as DHA. The industrial production of C. cohnii is already well established for the production of a DHA-rich additive to infant formula, this is mainly due to the fact that C. cohnii is able to produce a very specific fatty acid profile (mostly DHA). In aquaculture the main species of algae in hatchery and early feeding stages are autotrophic (they require light). Species such as I. galbana and P. tricornutum are commonly used(2)(3). These species are used as feed either for zooplankton species (rotifer and artemia – feed or enrichment) or directly to the juveniles, and are valued for many reasons not least of which are their rich content of fatty acids (primarily EPA and DHA)(2)(3). There are major disadvantages for aquaculturists in the production of these organisms; extensive infrastructure needs to be setup in order to cultivate these algae, resulting in additional infrastructural costs as well as additional labour. It would be immensely beneficial if the amount of autotrophic algae required in aquaculture could be reduced or eliminated entirely. This would result in a reduction of the costs encountered during hatchery production. A promising alternative to autotrophic species of microalgae are heterotrophic species such as the thraustochytrids, in particular Schizochytrium species. Heterotrophic species of microalgae differ in a number of ways from autotrophic microalgae, namely, they require an organic carbon source, such as glucose or more industrially available materials such as fruit processing waste or waste glycerol. They also require an aerated medium (an oxygen source) instead of the carbon dioxide required in autotrophic species cultivation. Due to the more energy rich medium that heterotrophic species are grown on and a lack of the need to photosynthesize, they are able to reach much higher cell densities in a shorter time than autotrophic microalgae. By adapting the

cod. There are currently no autotrophic microalgae that are capable of producing polyunsaturated fatty acids (PUFAs) feasibly(6). The product is easy to use and store. The existence of this company is testament to the fact that heterotrophic microalgae can be produced on a large scale and at a profit. Some benefits of using these heterotrophic microalgae in place of or as a supplement to autotrophic algae are as follows: • A better polyunsaturated fatty acid(PUFA) profile of the fed species (rotifer. they extract oil very rich in DHA for use in infant nutrition products. The most famous of which is Martek’s DHASCO(TM) process(8). Heterotrophic microalgal products are also available to aquaculture. one such product is the Algamac line available from Aquafauna biomarine (www... artemia. Cultivating these species in higher latitude countries is also not feasible due to the lack of natural (and consequently free.. further simplifying downstream separation processes. In addition the technology required to cultivate these species’(autotrophic sp. Such is the benefit of these heterotrophic microalgae.aquafauna.) .com).) is still in its infancy.) light. These species are consequently the only species that are capable of producing omega oils economically(5)(6)(8). Unlike autotrophic algae that need to be produced as they are required. making the technology accessible and cost effective. • • • • Species of heterotrophic microalgae are already in production for a number of applications. and is available on demand. Martek cultivate C. • • • • • Elaborate equipment is required Live algae is often used for aquaculture applications – requiring much labour and constant production Very dilute cultures are grown (due to the mutual shading effect) Downstream processing is difficult Moderate fatty acid yields are achieved Heterotrophic microalgae overcome many of these difficulties by possessing many favourable characteristics namely. cohnii on a large scale (a total fermentation capacity of around 200m3 (8). The algamac products are spray dried microalgal products used to great advantage in aquaculture.cultivation process accordingly a very high lipid concentration can be reached(7). The technology required for their growth is well established and is available on a bulk scale. the algamac products are produced in the US and shipped around the globe. Some of the difficulties in cultivating autotrophic microalgae are summarised as follows. • • • They are easily cultivated Well established technology is required for their cultivation Very high biomass concentrations are achievable High biomass concentrations facilitate downstream processing A reliable and very controllable production is achievable An extremely high lipid content is achievable Depending on the species chosen a very narrow range of fatty acids can be produced..

Receiving and maintaining of a promising heterotrophic strain a. - Intended Experiments 1. It is the aim of this project to investigate whether such a process is implementable and beneficial to aquacultural activities in the country. poultry and airy feed supplements). There seems to be no focused research efforts towards the production of these species of algae. using simpler equipment. Protein composition c. Schizochytrium sp b. Crythetocodinium cohnii 2. Basic medium requirements . • • • • It will be financially favourable to produce PUFA rich heterotrophic microalgae in Norway The production of such algae will help to reduce the labour and costs associated with the production of autotrophic algae There will be a marked improvement in the health and survival of aquacultured species partially fed with PUFA producing heterotrophic microalgae The production of these microalgae can be achieved with a better yield. sports bars. infant formula. The intended experiments will be aimed towards building a robust “Industrial” or “White” biotechnological process. Less labour and less space than the conventionally produced autotrophic microalgae Digestion of PUFA rich algae will be easier and more ecologically sound that conventional enrichments supplied by industry • It is also hoped that after these experiments are completed a further step will be taken towards the establishment of biotechnological processes in Northern Norway. for the production of mid – high value products at higher volumes than typical pharmaceutical style biotechnological processes.• • • • A more sustainable source of PUFA. an aquaculture intensive country. in contrast to fish oil Lower toxin concentration in contrast to fish oil – due to it being cultivated under controlled conditions A reliable supply throughout the year Easily stored and processed This new algal source is not yet produced in Norway. enriched vegetable oils) agricultural animal feeds (pet food supplements. additives for a variety of functional foods (breads. Fatty acid composition b. although the expertise exists. Potential spin off products from this project may include: a vegetarian omega oil (DHA) supplement. It is hoped that the intended experiments will show that. Flask culture of the above strain and baseline analysis of composition a.

Establishment of Pilot scale production media and operational protocol b. potato waste. Further medium optimisation 4. molasses. Large scale cultivation of species a.3. Provide samples for animal testing Equipment currently available for the experiment: • • • Flask culture and scale up facilities Analytical facilities Pilot scale tank Equipment and consumables required for the experiments • Medium components (from 250ml – 300L) o o o o Glucose Corn steep liquor Peptone Artificial seawater salt solution (trace elements and minerals) Industrial media (dairy waste. aquaculture waste streams & stickwater) o • Scale up level consumables o o o Silicon tubing Air filters (1micrometer) Tube clamps • Pilot scale Equipment and consumables o o o o Industrial agitator (for 300L volume tank) Sealing for tank Gas spargers Medium sterilisation equipment  Stainless steel tank (5-10L) Steam generator / electric heater Cooling system   o Gas compressor . waste glycerol. Mid-scale cultivation of species a.

o o o o Medium pump Basic separation equipment (freeze drying / spray drying / vacuum drying) Nitrogen gas Basic tubing and mounting for tank Schematic overview of the process: Figure 1: Basic Schematic of the experimental procedure Benefits felt from the project: • • Potential new and cost effective source of feed to hatcheries Potential new additive in formulated fish feed Potentially new bioremediation process (conversion of aquacultural wastewater to valuable byproducts) Potential new additive in human food supplements – Omega Research centre. Sintef. Oslo • • .

tissue culture. 2005 . S.K. ensuring a quick transition from research to market References: 1. Pilot scale fermentation of marine algae (cutting edge) Analysis of new sources of valuable biological products o o • Provide a product ready for use in industry. Food and Agricultural Organisation of the United Nations Regional office for the Asia Pacific. A.H. Place. F.Grima. Bar. Strain T66: effects of N and P starvation and =2 limitation” A. “Recovery of microalgal biomass and metabolites: process options and economics” E. 2008 8. bioremediation. “Biosynthesis and Regulation of Microbial Polyunsaturated Fatty Acid Production” M.D. Belarbi. I.” M.R. “Single Cell Oils” Z. Erik Lindebo and Derek Staples. Brown. Jakobsen. EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs. Medina. “Advanced DHA. HArel. Maseda. Josefsen. R. Certik. S. Otero 5.” Simon Funge-Smith.R. P. Ratledge.• Establish new Biotechnological techniques in Bodö namely: o Bubble column technology – very flexible technology used in. Bangkok.. A. Aasen. Zohar.Ferreira. Stubblefield.G. I Lein.A. K. J. A. Cohen. Dunstan.J. A. Shimizu. “Accumulation of docosahexanoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. “Asian Fisheries Today: The Production and use of Low Value/Trash Fish from Marine Fisheries in the Asia Pacific Region. C. pharmaceutical research. Volkman. M. Fabregas.W. J. Koven. J. 2001 3. 2005 2.A Fernandez. Jeffery.R. 1997 4. N. “Enriching Rotifers with “premium” microalgae Isochrysis aff. Y Chisti. Y. Y. A. Ström. Behrens.. G. 1998 6. E. “Nutritional properties of microalgae for mariculture” M. Galbana clone T-ISO” M. W. 2002 7.M.