Miguel G. Guerrero del Instituto de Bioqiímica Vegetal y Fotosíntesis de la Universidad de Sevilla-CSIC, presenta el mercado de producción de Bioethanol de microalgas y las ventajas de usar microalgas a la hora de producir BIoethanol. 8_04_2010

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

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

4. World ethanol production
eBIO: European Bioethanol Fuel Associations

5. 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

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

7. 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.

8. 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?

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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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)

18. 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

19. 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?

20. 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

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

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

23. 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)

24. 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

25. 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

26. 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