Biodiesel From Microalgae

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1 Biodiesel From Microalgae Lipid synthesis in microalgal cultures Shuo Yao Jingquan Lu Anders Brandt Claes Gjermansen Klaus Breddam

2 Oil yield l/ha/year Soybean 400 Sunflower 1000 Jathropha 2000 Oil Palm 6000 Algae 9000 Algal physiology and lipid synthesis Large scale growth Harvesting Extraction Processing

3 Commercial product Market size t/year Sales volume million $US/year Biomass Health Food Aquaculture Animal feed 300 Polyunsaturated FA ARA 20 DHA PFFA Extracts 10 Antioxidants beta-carotene Tocopherol 100 Coloring substances Astaxantin 300 (bm) 150 Phycocyanin 10 Phycoerythrin 2 Ferilizars/soil conditioners 5000 Ref:Pulz and Gross (2004), Spolaore et al (2006),Metting and Pyne (1986) 5572

4 Algal Oil production Costs Can Be Reduced by Integration with Wastewater Treatment Inspired by T.J. Lundquist, I.C. Woertz, N.W.T. Quinn, and J.R. Benemann: A Realistic Technology and Engineering Assessment of Algae Biofuel Production

5 System productivity Water quality Species control; robust local strains Low cost harvesting Biomass with high lipid content Productivities near the efficiency limits of photosynthesis

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7 The production facilities on which some of the assumptions are based Figure 1-1. Commercial microalgae production in open raceway paddlewheel mixed ponds. Left: Earthrise Nutritionals, LLC, California. Spirulina production, Ponds ~ 1 acre. Right: Cyanotech Co., Hawaii, producing Haematococcus pluvialis (red ponds) and Spirulina. Figure 1-2. Test facility for algae biomass production integrated with wastewater treatment Left: High Rate Ponds at Univ. of California, Berkeley. Right:The Roswell Algae Test Facility, New Mexico 7

8 Diatoms Algal species ~100,000 species; fresh and sea water Silica cell walls Store carbon as Lipids Green algae ~8,000 species; fresh and sea water Store carbon as Starch Accumulate lipids upon stress A few thousand strains are kept in culture collections throughout the world, A few hundred are being investigated for their chemical content and A handful are cultivated on an industrial scale.

9 Challenges Why so few? Difficult to domesticate Difficult to scale up from laboratory to large-scale cultivation Low harvest efficiency Production costs Culture medium, evaporation and rain dilution

10 HUNTLEY and REDALJE Mitigation and Adaptation Strategies for Global Change (2006)

11 Algae species Laboratory scale, max 2 litre Salt water Dunaliella sp Brackish water Nannochloropsis sp (constitutive lipid synthesis) Fresh water Chlorella sp Chlamydomonas sp Neochloris sp (constitutive lipid synthesis) Haematococcus sp Botryococcus sp

12 Algal Lipids Non-polar lipids Acyl lipids Phospholipids Polar lipids Glycolipids Non-acyl lipids (isoprenoids, carotenoids)

13 Triacyl glycerides (TAG) yield Two parameters to manipulate Cell size. (C. reinhardtii can range from 2 μm diameter to 12 μm in haploid wild type to twice this size in diploid strains) Cell number. Mitotic doubling rate; Cells per unit culture is determined by the density at which the cells shift into stationary phase. Little is known about why algae go into stationary phase at species-specific cell densities; Depletion of nutrients, buildup of toxic components in the media, quorum effects, and/or other genetic constraints. Nitrogen starved cells with lipid bodies

14 Where is TAG made and stored Radakovits et al. 2010, eukaryotic cell

15 Lipid synthesis induced by stress Chlorella vulgaris Haematococcus pluvalis Synthesis of astaxantin Neochloris oleoabundans Dunaliella salina SG medium: 1: - N -P 2: -P 3: -N 4: complete

16 Genetic Toolbox Ability of cells to grow on agar plates Identification of selectable markers Development of transformation vectors. Transformation methods (Nuclear and chloroplast). Sexual crossing (breeding). Homologous recombination/ gene replacement vs. random insertion.

17 Beneficial Characteristics Efficient in biomass production Programmed production of fatty acids and lipids High fatty acid content Fatty acids easily released Cell debris easily recovered Combined with waste water treatment and industrial CO2 removal

18 UV mutagenesis Search for cell cycle division mutants UV intensity: 4000 µj/cm 2 Light intensity: µJ/cm 2 Permissive temperature: 25 degrees Restrictive temperature: 34 degrees

19 0 UV 22 degrees UV 4000 µj/cm 2

20 CC Plate screening 20 Præsentationens titel

21 WT137C (mt+) -34 WT137C (mt+) Præsentationens titel

22 M37-22-day0 M37-22-day0 22 Præsentationens titel

23 M37-34-day2 M37-34-day2 23 Præsentationens titel

24 MI2-34-day2 MI2-34-day2 24 Præsentationens titel

25 UV mutagenesis Search for cell cycle division mutants UV intensity: 4000 µj/cm 2 Light intensity: µJ/cm 2 Temperature: 25 (permisive), 35(restrictive) 7500 colonies screened, survival rate: 37% average 15 TS mutants found, 0.2% of screened colonies 4 TS mutants are Giant cells, 27% of TS mutants

26 Future algae oil production? Biomass production Lipid formation Raceway Ponds Induction Residual products Water Lipids Debris

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