Bioethanol from microalgae

Bioethanol from microalgae? Miguel G. Guerrero Instituto de Bioquímica Vegetal y Fotosíntesis Universidad de Sevilla Consejo Superior de Investigaciones Científicas Sevilla, Spain

Total EU27 biodiesel production for 2008 was over 7.7 Mton (~8,600ML) EEB: European Biodiesel Board

World ethanol production eBIO: European Bioethanol Fuel Associations

EU Ethanol production (ML) EU MEMBER STATE 2008 2007 2006 2005 2004 Austria 89 15 Belgium 51 Czech Republic 76 33 15 Finland 50 13 3 France 950 539 293 144 101 Germany 581 394 431 165 25 Hungary 150 30 34 35 Total imports of Ireland 10 7 bioethanol in EU: Italy 60 60 128 8 1900 ML in 2008 Latvia 15 18 12 12 12 Lithuania 21 20 18 8 Netherlands 9 14 15 8 14 Poland 200 155 120 64 48 Slovakia 94 30 Spain 346 348 402 303 254 Sweden 78 120 140 153 71 eBIO: European Bioethanol UK 75 20 Fuel Associations TOTAL 2855 1803 1608 913 528

Raw materials for ethanol production in Europe (2008) eBIO: European Bioethanol Fuel Associations

Microalga Eukaryotic microalgae and prokaryotic cyanobacteria are the major representatives of oxygen- evolving photosynthetic microorganisms COLLECTIVELY REFERRED TO AS MICROALGAE U.S. Department of Energy Genome Programs http://genomics.energy.gov.

Claimed advantages of microalgae over crop plants for biofuel production • Faster growth • Higher productivity • Use saline, brackish, waste water • Do not compete with food/feed agriculture • Can have very high carbohydrate/oil content • Lower water consumption? • Lower costs of production/processing?

Ethanol yields for various crops CROP PRODUCTIVITY (liters per hectare) Wheat 2,500 Corn 3,500 Sugar beet 6,000 Microalgae (projection) 20,000

Products from microalgae Biomass Pigments (phycobiliproteins, carotenoids) Essential fatty acids (long-chain PUFAs) Bioactive compounds (diverse chemical nature and biological activity) Exopolysaccharides Major cell components (triglycerides, starch, glycogen) as feedstock for biofuels (biodiesel, bioethanol) Simple molecules with high energy content Ammonia Hydrogen Alcohols Fatty acids

Biofuel generation from CO2 Through photosynthesis, at the expense of sunlight energy, energy-rich compounds are synthesized from oxidized, low energy substrates. The generation of an organic fuel entails besides CO2 removal CARBOHYDRATES ALCOHOLS H2 LIPIDS -0.4 V Fd HYDROCARBONS H+ CO2 e +0.8 V H2O THYLAKOIDS O2 LIGHT

Choosing the microalga for producing bioethanol’s feedstock Factors to be considered in the selection Growth rate (µ); productivity (P= µ·Cb) Selective advantages: tolerance to temperature, pH, and radiation extremes; secretion of allelopatic metabolites; ability to fix N2 High yield in fermentable carbohydrates (starch, glycogen, EPS?) Easy (cheap) harvesting

Microalgae as potential source of carbohydrates Strain of Chlorella Carbohydrates (% of dry weight) +N -N C. ellipsoidea SK 15,0 21,0 C. pyrenoidosa 82 24,0 37,3 C. pyrenoidosa 82T 31,8 67,9 C. pyrenoidosa TKh-7-11-05 10,0 44,2 C. sp. K 18,4 54,5 C. vulgaris 157 10,3 44,0 Data from Vladimirova et al (1979) & Zhukova et al (1969) in Soviet Plant Physiology

Cyanobacteria as potential source of carbohydrates (Vargas et al. 1998, J. Phycol. 34, 812) Strain Carbohydrates (% of dry weight) ________________________________________________ Anabaena sp. ATCC 33047 28.0 ± 2.0 Anabaena variabilis 22.3 ± 2.5 Anabaenopsis sp. 16.3 ± 1.5 Nodularia sp. (Chucula) 16.9 ± 2.6 Nostoc commune 37.6 ± 2.5 Nostoc paludosum 26.6 ± 1.9 Nostoc sp. (Albufera) 26.8 ± 4.0 Nostoc sp. (Caquena) 23.3 ± 1.7 Nostoc sp. (Chile) 23.3 ± 2.0 Nostoc sp. (Chucula) 15.7 ± 1.8 Nostoc sp. (Llaita) 20.2 ± 1.5 Nostoc sp. (Loa) 32.1 ± 1.2

Marine strain of Anabaena (ATCC 33047, CA) • High rate of CO2 fixation into organic matter • High productivity • No requirement for combined N (N2-fixer) • Easy harvesting • Wide tolerance to: • temperature (optimum 40ºC; 30-45) • pH (optimum 8.5; 6.5-9.5) • irradiance • salt • Carbohydrate content: 23-34% of dry biomass in actively growing cultures

Simultaneous to growth and biomass increase, Anabaena sp. ATCC 33047 releases to the medium substantial amounts of an exopolysaccharide (EPS) The EPS exhibits interesting rheological properties, and contributes to easy harvesting of biomass The EPS can find different applications, including fermentation

Anabaena cultures outdoors PRODUCTIVITY 0.05–0.6 g organic matter (biomass+EPS) L-1 d-1 equivalent to 0.1–1.0 g CO2 fixed L-1 d-1 YIELD OF FLAT PANEL REACTOR 0.1 (winter) to 0.35 (summer) g biomass L-1 d-1= ~35 g biomass m-2 d-1(8-11 g carbohydrates m-2 d-1)

ELECTRICITY POWER PLANT POWER PLANT FLUE FOSSIL FUEL FOSSIL FUEL (combustion) (combustion) PURIFICATION PURIFICATION CO2-RICH GAS GASES SUNLIGHT SUNLIGHT HEAT PHOTOBIOREACTOR PHOTOBIOREACTOR (INOCULATED CULTURE) (INOCULATED CULTURE) BIOMASS BIOMASS DIVERSE ± OTHER ORGANIC COMPOUNDS ± OTHER ORGANIC COMPOUNDS APPLICATIONS

Establishing a production process for microalgae as source of bioethanol’s feedstock Factors to be considered (and optimized) • Organism - natural isolate (production site) - strain from culture collection - carbohydrate overproducing mutant (?) • Culture system - open, closed, semi? • Operating conditions - batch, semi-continuous, continuous? - nutrient limitation(s)? - one-stage, two-stage?

A plausible (although ambitious) objective, considering present state of art (high insolation area) • Reactors of ~50 L m-2 operating at mean volumetric productivity of ~0.7 g biomass L-1 day-1 (or of 140 L m-2 at 0.25 g L-1 day-1). Productivity = 35 g biomass m-2 day-1 • For a carbohydrate content of ~30% = 10.5 g carbohydrate m-2 day-1 • Surface extrapolation = 0.35 ton biomass (0.105 ton carbohydrate) ha-1 day-1 • Surface + time extrapolation (effective operation 300 days per annum) =105 ton biomass (31.5 ton carbohydrate) ha-1 year-1 ~(19,000 L ethanol) ha-1 year-1

Microalgal metabolic pathways that can be leveraged for biofuel production Radakovits et al. (2010) Eukaryotic Cell 9: 486-501

Starch metabolism in green microalgae Radakovits et al. (2010) Eukaryotic Cell 9:486-501

Fermentative production of bioethanol Raw materials • Sugar cane (Brazil) • Corn (USA) • Wheat, corn, sugar beet (Europe) • Alternatives: lignocellulosic materials; microalgae CO2 emissions Alcoholic fermentation (yeasts) C6H12O6 2 CH3CH2OH + 2 CO2 (16 kJ g-1) (30 kJ g-1)

Ethanol photoproduction from CO2 LIGHT 2 CO2 + 3 H2O → CH3CH2OH + 3 O2 CO2 fixation Ethanol photosynthesis CO2 Calvin cycle 3-PGA PYRUVATE ACETALDEHYDE ETHANOL PDC ADH

Synechocystis sp. PCC6803 (Sectionn I , Rippka et al., 1979) • Fast growth • Easy culture Model cyanobacterium Growth on glucose Full genomic sequence available (http://www.kazusa.or.jp) Transformable (chromosome and plamid) Homologous recombination

Strategy for obtaining Synechocystis strains able to synthesize ethanol 1. Insertion in Synechocystis genome of Zymomonas genes involved in ethanol synthesis through homologous recombination P pdc-adh Secuence homologous toSynechocystis DNA (needed for reombination) P Endogenous promotor (externally inducible) Pyruvate decarboxylase and alcohol dehydrogenase genes Antibiotic-resistance cassette 2. Analysis of proper integration in genome, and of full segregation, by Southern Blot 3. Expression analysis of genes in a single RNAm under inducing conditions, by Northern Blot 4. Measurement of enzyme activities in cell extracts under inducing conditions 5. Verification of ethanol presence in outer medium

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Bioethanol from microalgae

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