Optimizing Lipid Production by Planktonic Algae LIPIDO

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1 Optimizing Lipid Production by Planktonic Algae LIPIDO Kristian Spilling, Finnish Environment Institute, SYKE Project leader: Timo Tamminen

2 Potential benefits of algae Algae can be grown in a way that do not compete for fertile land Has an advantageous energy / carbon balance Salt or wastewater can be used as the base for culturing Can be coupled with CO 2 producing industry High growth rate and lipid yield

3 Technology (product and production method) Liquids vs. gas (biogas /methane vs. hydrogen) Biodiesel vs. ethanol (alcohol) or both Closed vs. open systems (or hybrid) Closed GreenFuel, Algenol Hybrid (Shell) Open Most existing plants today Harvesting vs. milking Harvesting GreenFuel Milking - Algenol

4 LIPIDO Nordic Energy Research Northern European Innovative Energy Research Programme (N-INNER) Project duration: Project budget approx.: Partners: Finland 2 Norway 2 Germany 1 Iceland 1

5 Finland Finnish Environment Institute (SYKE) Timo Tamminen, Kristian Spilling, Jukka Seppälä Algal growth experiments, environmental parameters VTT, Technical Research Centre of Finland Niklas von Weymarn, Kirsi-Marja Oksman- Caldentey, Heiko Rischer Lipidomics, overall biorefinery concept, downstream and sidestream processing

6 Norway Norwegian University of Science and Technology (NTNU), Olav Vadstein, Yngvar Olsen Algal stoichiometry, growth yield and reactor technology University of Oslo Tom Andersen, Per Færøvig Algal growth response and optimization

7 Germany Ludwig Maximilian University (LMU) Herwig Stibor, Maria Stockenreiter Algal stoichiometry and fatty acid composition

8 Iceland Iceland GeoSurvey Thráinn Fridriksson, Kristín Kroyer Geothermal and industrial CO 2 sources, energy development

9 Project portfolio Additional Finnish projects (SYKE/VTT): ALGIESEL (Finnish Academy/ SusEn) MICROFUEL (Tekes / BioRefine) Additional Norwegian projects Funded by the Research Council of Norway

10 LIPIDO Project goals (and ALGIESEL) Screening the most promising algal species from temperate environments Optimizing their growth and lipid yield as functions of growth condition Testing the practical applicability of coupling algal culturing to CO 2 emission Screening commercially interesting by-products from biomass of selected species Proof-of-concept

11 MICROFUEL: VTT & SYKE (Tekes) Model factory Potential of using remaining algal biomass Yeast production of lipids Light CO 2 / Air Water Nutrients Algae AP CR LS Lipids Total mass Dry mass Ethanol or methane production Liquid phase => What to do? Biomass residue => What to do? AP = algae production, CR = cell recovery, LS = lipid separation

12 Activities 2008 Start January 2008 Screening Bi-products (ethanol) Harvesting (flocculation) Lipid optimization

13 Biomass production (approx 8 kg FW = 2 kg DW)

14 Harvesting A lot of water need to be removed Few grams DW / L -1 Estimated to be % of total production cost 1 1) Gudin and Therpenier 1986

15 Screening different species Media adjusted to be N limited when entering the stationary growth phase ~15 species screened Biomass max max / 2 sampling Time

16 Lipid formation Light and CO2 Growth algae Nutrients Storage

17 Lipid formation Light and CO2 Growth algae Nutrients Storage Lipids Carbohydrates

18 Screening different species FAME FFA mg / g FW SH log SH stationary CW log CW stationary TB log TB stationary SC log SC stationary MA log MA stationary GYM stationary PT stationary SH = Scrippsiella hangoei, CW = Chaetoceros wighamii, TB = Thalassiosira baltica SC = Skeletonema costatum, MA = Melosira arctica, GYM = Gymnodinium sp. PT = Pauliella taeniata

19 % FFA vs. FAME 100% 80% 60% 40% 20% SH log SH stationary CW log CW stationary TB log TB stationary 0% SC log SC stationary MA log MA stationary GYM stationary PT stationary FFA FAME SH = Scrippsiella hangoei, CW = Chaetoceros wighamii, TB = Thalassiosira baltica SC = Skeletonema costatum, MA = Melosira arctica, GYM = Gymnodinium sp. PT = Pauliella taeniata

20 Increase from log to stationary growth phase SH CW TB SC MA FAME FFA SH = Scrippsiella hangoei, CW = Chaetoceros wighamii, TB = Thalassiosira baltica SC = Skeletonema costatum, MA = Melosira arctica

21 Department II, Aquat. Ecology, LMU- München Maria Stockenreiter, Martin Steinböck, Florian Haupt, Herwig Stibor

22 Semibatch cultures Nitrogen limitation resulted in cell specific lipid content increase Fluorescence per cell N+/ 100µM photons N+/ 30µM photons N-/ 100µM photons N-/ 30µM photons 1000 Botryococcus braunii Chlamydomonas reinhardtii Staurastrum tetracerum Anabaena cylindrica Nile Red stain, 1000 fluorescence units = 11pg lipid Mechanism: stop of cell division and accumulation of photosynthetic products in the form of lipids

23 Semibatch cultures Problem: low biomass due to nutrient limitation 1,00E+09 1,00E+08 N+/ 100µM photons N+/ 30µM photons N-/ 100µM photons N-/ 30µM photons Tot.Vol. (fl/ml) 1,00E+07 1,00E+06 1,00E+05 1,00E+04 Botryococcus braunii Chlamydomonas reinhardtii Staurastrum tetracerum Anabaena cylindrica

24 Growth factors affecting lipid content Experimental design: Chemostat coupeld with semibatch culture: algae were cultivated in first a WC- nutrient solution (chemostat) and second in a modified WC-nutrient solution with reduced nitrogen content (semibatch culture). light conditions: 100 µm photons and 12 h light cycle. experimental time: 30 days. algal species: Botryococcus braunii, Staurastrum tetracerum peristaltic pump

25 Measurements: total-biovolume of algae (Cell- Counter). lipid content by fluorescence measurement of cells stained with Nile Red (FlowCam).

26 Two stage set-up Semibatch cultures Chemostat coupled with semibatch Botryococcus braunii Staurastrum tetracerum Botryococcus braunii Staurastrum tetracerum 1,00E+09 1,00E+08 1,00E+08 Tot. Vol.(fl/ml) 1,00E+07 1,00E+06 1,00E+05 Tot. Vol.(fl/ml) 1,00E+07 1,00E+06 1,00E+05 1,00E+04 N high N low 1,00E+04 stage1 (N high) stage2 (Nlow)

27 Two stage set-up Semibatch cultures Chemostat coupled with semibatch Fluorescence per cell Botryococcus braunii Staurastrum tetracerum Fluorescence per cell Botryococcus braunii Staurastrum tetracerum 1500 N high N low 1500 stage1 (N high) stage 2 (N low) 1000 fluorescence units = 11pg lipid

28 Conclusion for lipids Nitrogen reduction increased lipid production of different algal species. Highly species specific Botryococcus braunii and Scrippsiella hangoei had the highest cell specific lipid content, Two stage culturing system resulted in higher nutrient specific biomass production and lipid content of algae compared to a one stage culturing system

29 Challenges ahead Lowering production cost Getting high enough production and lipid yield Water handling Managing cultivation Harvesting Having realistic targets There are some overoptimistic calculations of the potential Agriculture has been developed for millennia, algal cultivation has a ~50 year history

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