Oils from micro-algae: achievements and prospects

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1 Oils from micro-algae: achievements and prospects Colin Ratledge University of Hull, UK

2 Single Cell Oils

3 SCOs are triacylglycerols CH 2 O-CO-(CH 2 ) XCH 3 CHO-CO-(CH 2 ) Y CH 3 CH 2 O-CO-(CH 2 ) z CH 3 glycerol fatty acids These are the same as are present in all major plant oils: e.g. soybean oil sunflower oil rapeseed (Canola) oil palm oil, etc. The fatty acids are also the same as those in plant oils

4 Sources of single cell oils (the oleaginous microorganisms) Algae complex lipids associated with photosynthesis but 20-50% neutral lipids Fungi can contain up to 60-70% oil Yeasts some species >70% oil Bacteria produce poly-betahydroxybutyrate and related polymers; one or two species will produce triacylglycerols

5 Microbial technology for lipids Cultivation of ALL microorganisms is expensive even algae growing photosynthetically. Algae are being used to produce fatty acids for biodiesel: economically feasible? Fatty acids for biodiesel: ideally palmitic acid (16:0) plus oleic acid (18:1) Algae grown photosynthetically have wide range of fatty acids and many polyunsaturated ones.

6 Alternative uses for fatty acids Nutritional and or medical foods Target fatty acids: the polyunsaturated fatty acids (PUFAs) Arachidonic acid (ARA): 20:4 n-6 Eicosapentaenoic acid (EPA): 20:5 n-3 Docosahexaenoic acid (DHA): 22:6 n-3 Already considerable demand for oils containing these fatty acids (>5000 tonnes/year): selling prices up to $80-100/kg Fish oils: good source of EPA + DHA but not suitable for infants; also not suitable for vegetarians and some religious groups.

7 The process of SCO production Essential for there to be excess carbon (C0 2 or glucose) AND a nutrient limitation to stop cell proliferation

8 Gluc os e/nitrogen (arbitrary values) Biomass (dry wt) Lipid (% dry wt) Oil accumulation process in an oleaginous microorganism 100 balanc ed growth lipid ac cum ulation biomas s nitrogen lipid glucos e Time (arbitrary s cale) 0

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11 Algae for Oils Huge commercial interest in algal oils for biofuels (biodiesel) since 2001 Over 200 start-up companies Aim to produce algal biodiesel cheaper than petroleum oil Concept: use photosynthesis to produce lipids in selected micro-algae Major investments and US government grants + tax relief and incentives

12 Algae for key lipids (microalgae only being considered) Obligate phototrophs can only grow photosynthetically Facultative phototrophs can grow either photosynthetically or with fixed carbon source (e.g. glucose) Mixotrophs can grow photosynthetically AND with a fixed carbon source Obligate heterotrophs can only grow with a fixed carbon source (glucose, sucrose etc.)

13 Lipids from obligate phototrophs or algae grown photosynthetically Cyanobacteria (blue-green algae): e.g. Spirulina but low content (<10%) lipid. (But good for proteins) Botyrococcus braunii: 25-75% lipid but mainly complex branched hydrocarbons; slow grower Chlorella spp.: up to 60% lipid Haematococcus pluvialis: 30-40% lipid but used commercially to produce astaxanthin Isochrysis galbana: 22-38% lipid Nannochloropsis sp.: 30-68% lipid Nitzschia spp.: ~45% lipid Parietochloris incisa: 30-45% lipid Phaeodactylum tricornutum: 20-57% lipid Scenedesmus obliquus: 50% lipid

14 BUT

15 Lipids from photosynthetically-grown algae are complex materials and require hydrolysis to recover fatty acids whatever their use. Almost all data on lipid productivity are derived from photobioreactors (NOT outdoor cultivation) using constant temp + CO 2 addition and often constant illumination. The Dark Reaction (respiration and metabolism of stored oils) during the night is largely ignored. Photobioreactors are too expensive to use for production of a cheap biofuel.

16 PBRs in Israel today!

17 PROBLEMS WITH ALGAL CULTURE Supply of CO 2 source and addition Supply of phosphate and nitrogen Temperature day/night; summer/winter Water fresh water or seawater? Water evaporation how is it to be replenished? If seawater is used, fresh water must be added to prevent salt buildup. Land: must be of very low value

18 CO 2 Theoretically: 3 tonnes CO 2 needed to synthesize 1 tonne triacylglycerol (without producing any cells). Highest conversions attained in closed photobioreactors but impractical for biofuels. In practice in outdoor cultivation: 14 tonnes CO 2 used to produce 1 tonne oil. Improvements needed for CO 2 dispersal. Use waste flue-gases? Will need clean-up. But limited availability in right locations for algal cultivation.

19 Supply of N, P and Water To produce 10% of the US liquid fuel using algal oils would consume more than the entire US supply of nitrogen and phosphate. (Ronald Pate: Applied Energy 2012, 88:3377) To produce 5% of US fuel from algal oils would consume up to 123 million tonnes of freshwater = 10% of current water consumption. (National Research Council of USA: Report October 24 th, 2012)

20 National Research Council Report (Sustainable Development of Algal Biofuels in the USA) To be sustainable, algal biofuels would need to produce more energy than is required in their production. AIM: 3 x Energy out = Energy in Current yields are about 0.13 and maybe >1. Conclusion: algal biofuels (produced by current technologies) will place unsustainable demands on energy, water and nutrients. Little immediate prospect of economically-viable algal biofuels.

21 Likely productivities and costs (from Cohen & Ratledge, Lipid Technol vol. 20) Alga considered: Pleurochrysis carterae with 33% oil content being grown in raceway ponds Best productivity based on outdoor cultivation = 22 tonnes oil per hectare/year Calculated cost of oil = $US 21,000/ton or about $ 3000 per barrel. (Petroleum oil ~ $100/barrel) Productivity would have to be 110 tonne oil/ha.yr; costs = $800/tonne = $115/barrel.

22 Sapphire Energy Inc. Using phototropically growing microalga (possibly Chlorella?) Location: New Mexico using raceway ponds Cost of facility: $60 million Target: 10,000 barrels oil/day by 2018 Aim: biomass with 30% oil (HOW?) and to achieve 37 tonne/hectare Production costs: $16000/tonne = $2300/barrel but consider that $ /t is possible.

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25 Current consensus of opinion suggests that the cost of producing algal oil will be at least $5600-$7000/tonne ($ barrel) for the foreseeable future. Not surprisingly, most algal companies are now refocusing on higher value products or using a different strategy

26 Solazyme Inc. using Chlorella but growing heterotrophically using fixed carbon (glucose, sucrose or mixed sugars) as feedstock Large-scale demonstration facility in Illinois; plans to use fermenters up to 500 m 3 by 2015 Will still qualify for US Tax Relief as using a cultivated alga

27 Chlorella (typical green alga)

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29 Chlorella protothecoides grown heterotrophically on glucose (from Xiong et al. Appl. Microbiol. Biotech :29-36) Cell density: >50 g dry wt/l in 7 days Lipid content of cells: 50% (w/w) Fatty acid profile (rel. % w/w): 16:O 10 18: : :2 19 (When grown photosynthetically >80% fatty acids are unsaturated or polyunsaturated.)

30 Production of omega-3 polyunsaturated fatty acids Targets: DHA (22:6 n-3) and EPA (20:5 n-3) BUT not together. Uses: infant nutrition; human nutrition and also as medical foods or (for EPA) for clinical treatment of hypertriglyceridaemia. Mixtures of EPA + DHA from fish oils for human nutrition (prevention of heart diseases) but not acceptable to vegetarians etc. Fish oils production: >100,000 t/yr but increasing at 8000 t/yr; +95% of market.

31 DHA [Docosahexaenoic acid: 22:6 (n-3)] Requirement is for a DHA-only oil; EPA not to be present. Fish oil fractionation is too expensive. Not available from plants or other sources. Few algae produce such an oil: but 2 exceptions.

32 Schizochytrium sp. thraustochyrid alga, marine or euryhaline, unicellular, obligate heterotroph

33 Production of DHA using Schizochytrium sp. and related thraustochyrids Probably the fastest growing oleaginous microorganism Grown heterotrophically using glucose in fermenters m 3. Can reach 200 g/l in 3 days. Operational cell density may be lower. Developed by: OmegaTech Inc, Boulder, Colorado. Company taken over by Martek in 2002; now part of DSM. Several other companies using similar organisms to produce DHA oils. Production: probably <200 tons oil/year. Uses: Nutritional supplement for adults. Used in infant formulas in China. Could be used for fish feeding but requires cell fracture to allow uptake of DHA. Safety: Oil used in extensive animal trials and with human volunteers. Zero toxicity. FDA approval. GRAS status.

34 Schizochytrium sp.: DHA-SCO Oil content: >50% (w/w) of cell biomass Fatty acids %(w/w) in oil 14:0 8 16: :1 to 20:3} 3 20:4(n-3&n-6) 2 20:5 (EPA) 2 22:5(n-6)* 17 22:6(n-3)** 41 *DPA - unusual fatty acid, but found in human brain tissue. Not harmful - but does not count towards the DHA content of the oil. ** DHA Route of synthesis of PUFAs via polyketide synthase

35 Crypthecodinium cohnii dinoflagellate, marine, unicellular, flagellated, obligate heterotroph

36 Production of DHA using Crypthecodinium cohnii Grown heterotrophically using glucose as carbon and energy source. Time of growth: 8-10 days; Cell dry wt: ~60-80 g/l; lipid content: ~40% Developed by: Martek BioSciences Inc, USA in Large-scale production began in 1995/6. (Now owned by DSM) Production: ~ tonnes of oil/year using m 3 fermenters. Uses: nutritional supplement for infant formulas. Mixed with arachidonic acid (from Mort. alpina). Sold in ~70+ countries. Safety: rigorous testing in animals and human volunteers. Zero toxicity. FDA approval in Generally Recognized As Safe status (GRAS).

37 Oil content: Crypthecodinium cohnii: DHA-SCO 40% w/w of the dry cells Fatty acids %(w/w) in oil 14: : :1 2 18:0 <1 18: :2 <1 22:6 (DHA) 40 No intermediate fatty acids between 18:1 and DHA due to unusual biosynthetic pathway.

38 EPA [Eicosapentaenoic acid (20:5 n-3)] Requirement for an EPA-only oil for specific clinical applications. Currently produced by Amarin and GSK using (very expensive) fish oil fractionation.

39 Algae for EPA Grown photosynthetically (in photobioreactors with CO 2 enrichment) Isochrysis galbana: 24 mg EPA/L per day Monodus subterraneus: 59 mg/l.day Phaeodactylum etricornutum: 34 mg/l.day Porphyridium cruentum: 4 mg/l.day ****************************** Grown heterotrophically with glucose Nitzschia laevis: 175 mg/l.day (In all cases, complex lipids produced requiring hydrolysis, extraction and re-esterification.) (from Wen & Chen 2010 in Single Cell Oils)

40 Rival process to algal technology for EPA EPA-rich oil produced by GM-yeast (Yarrowia lipolytica) grown on glucose. >55% total fatty acids as EPA; little DHA or ARA. Developed by DuPont, USA. Costings: unknown Applications: human nutrition as a neutraceutical. Medical food?

41 Algal Oils with EPA + DHA Wide range of possibilities using photosynthetic cultures Chlorella spp. Dunaliella salina? Isochrysis galbana Nannochloropsis sp. Pavlova spp. Pinguiococcus Rhodomonas Oils will be used as neutraceuticals for people who do not want to consume fish or fish oils. Companies involved: Solaharvest (Canada/Arizona), Aurora Algae (Australia), Advanced Algal Technologies (links to China), Qponics (Australia), AlgalBio (Canada), BioProcess Algae (USA) and many others

42 Future Prospects for Algal Oils as biofuels Improved CO 2 fixation plants use CO 2 and accumulate oils; why not algae? Re-engineer photosynthesis to use light in the red and infra-red ( nm). (see Blankenship et al. Science 2011) Re-engineer to achieve lipid excretion: one tonne of cells could then produce many tonnes of oil.

43 Ideal production system for algal biofuels Sunlight CO 2 Cells grow and produce lipids Large algal cells Oil excreted outside cell (adapted from Wijffels & Barbosa, Science 2010)

44 Future Prospects for Algal Oils as neutraceuticals The next 5 years will see several (or many) algae being used for PUFA production. DHA already in production. DHA + EPA producible using various algae as alternative (but more expensive) to fish oils. EPA-only oil now feasible: Nannochloropsis, Nitzschia, Monodus etc. (Rival process using GMyeast technology). Prospects for GM-plants to produce EPA/DHA remain a distant hope. (Always in next 10 years!)

45 Algae for fish feeding? Whole biomass to be used. Some breakage of cell wall essential for digestibility. Current costs at least 4x price of fish meal. Needs government or EU legislation to stop fish being fed to fish as unsustainable. BUT realistic and cheap alternatives needed. Schizochytrium biomass probably the cheapest so far! And has given promising results in small-scale trials.

46 THANK YOU