BioethanolBioethanol fromfrom microalgaemicroalgae??

Instituto de BioquInstituto de Bioquíímica Vegetal y Fotosmica Vegetal y Fotosííntesisntesis

Universidad de SevillaUniversidad de SevillaConsejo Superior de Investigaciones CientConsejo Superior de Investigaciones Cientííficasficas

Sevilla, Sevilla, SpainSpain

Miguel G. GuerreroMiguel G. Guerrero

Total EU27 biodiesel production for 2008 was over 7.7 Mton (~8,600ML)

EEB: European EEB: European BiodieselBiodiesel BoardBoard

World ethanol productionWorld ethanol production

eBIO: European Bioethanol Fuel Associations

EU Ethanol productionEU 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

Ireland 10 7

Italy 60 60 128 8

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

UK 75 20

TOTAL 2855 1803 1608 913 528

Total imports of bioethanol in EU: 1900 ML in 2008

eBIO: European BioethanolFuel Associations

Raw materials for ethanol production Raw materials for ethanol production in Europe (2008)in Europe (2008)

eBIO: European Bioethanol Fuel Associations

U.S. Department of Energy Genome Programshttp://genomics.energy.gov.

Microalga

Eukaryotic Eukaryotic microalgaemicroalgae and and prokaryotic prokaryotic cyanobacteriacyanobacteria

are the major are the major representatives of oxygenrepresentatives of oxygen--evolving photosynthetic evolving photosynthetic

microorganismsmicroorganisms

COLLECTIVELY COLLECTIVELY REFERRED TO AS REFERRED TO AS

MICROALGAEMICROALGAE

Claimed advantages of Claimed advantages of microalgaemicroalgae over over crop plants for crop plants for biofuelbiofuel productionproduction

• 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 cropsEthanol yields for various crops

CROP PRODUCTIVITY(liters per hectare)

Wheat 2,500

Corn 3,500

Sugar beet 6,000

Microalgae (projection) 20,000

ProductsProducts fromfrom microalgaemicroalgaeBiomassBiomass

Pigments (Pigments (phycobiliproteinsphycobiliproteins, , carotenoidscarotenoids))

Essential fatty acids (longEssential fatty acids (long--chain chain PUFAsPUFAs))

Bioactive compounds (diverse chemical nature and biological actiBioactive compounds (diverse chemical nature and biological activity)vity)

ExopolysaccharidesExopolysaccharides

Major cell components (triglycerides, starch, glycogen) as feedsMajor cell components (triglycerides, starch, glycogen) as feedstock for tock for biofuelsbiofuels (biodiesel, (biodiesel, bioethanolbioethanol))

Simple molecules with high energy contentSimple molecules with high energy contentAmmoniaAmmoniaHydrogenHydrogenAlcoholsAlcoholsFatty acidsFatty acids

BiofuelBiofuel generationgeneration fromfrom COCO22Through 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

-0.4 V

+0.8 V

LIGHTLIGHT

THYLAKOIDSHH22OO

H+

H2

OO22

Fd

COCO22

CARBOHYDRATESALCOHOLS

LIPIDSHYDROCARBONS

e

Choosing the Choosing the microalgamicroalga for for producing producing bioethanolbioethanol’’ss feedstockfeedstock

Factors to be considered in the selectionFactors to be considered in the selection

Growth rateGrowth rate ((µµ); ); productivityproductivity (P= (P= µ·µ·CbCb))

Selective advantages: tolerance to temperature, pH, and Selective advantages: tolerance to temperature, pH, and radiation extremes; secretion of radiation extremes; secretion of allelopaticallelopatic metabolites; metabolites; ability to fix Nability to fix N22

High yield in fermentable carbohydrates (starch, glycogen, High yield in fermentable carbohydrates (starch, glycogen, EPS?)EPS?)

Easy (cheap) harvestingEasy (cheap) harvesting

MicroalgaeMicroalgae as potential source of as potential source of carbohydratescarbohydrates

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. pyrenoidosaTKh-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

CyanobacteriaCyanobacteria as potential source of as potential source of carbohydratescarbohydrates

(Vargas et al. 1998, J. Phycol. 34, 812)

Strain Carbohydrates(% of dry weight)

________________________________________________Anabaena sp. ATCC 33047 28.0 ± 2.0Anabaena variabilis 22.3 ± 2.5 Anabaenopsis sp. 16.3 ± 1.5Nodularia sp. (Chucula) 16.9 ± 2.6Nostoc commune 37.6 ± 2.5 Nostoc paludosum 26.6 ± 1.9 Nostoc sp. (Albufera) 26.8 ± 4.0Nostoc sp. (Caquena) 23.3 ± 1.7 Nostoc sp. (Chile) 23.3 ± 2.0Nostoc sp. (Chucula) 15.7 ± 1.8Nostoc sp. (Llaita) 20.2 ± 1.5Nostoc sp. (Loa) 32.1 ± 1.2

Marine Marine strainstrain ofof AnabaenaAnabaena(ATCC 33047, CA)

• High rate of COCO22 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, Anabaenasp. ATCC 33047 releases to the medium substantial amounts of an exopolysaccharide (EPS)

The EPS exhibits interesting rheologicalproperties, and contributesto easy harvesting of biomass

The EPS can find differentapplications, including fermentation

AnabaenaAnabaena cultures outdoorscultures outdoors

PRODUCTIVITYPRODUCTIVITY0.05–0.6 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 REACTORYIELD OF FLAT PANEL REACTOR0.1 (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)

FOSSIL FUELFOSSIL FUELPOWER PLANT (combustion)

POWER PLANT (combustion)

ELECTRICITYELECTRICITY

FLUE GASES

PURIFICATIONPURIFICATION CO2-RICH GAS

SUNLIGHTSUNLIGHT

PHOTOBIOREACTOR(INOCULATED CULTURE)PHOTOBIOREACTOR(INOCULATED CULTURE)

HEAT

BIOMASS± OTHER ORGANIC COMPOUNDS

BIOMASS± OTHER ORGANIC COMPOUNDS

DIVERSE APPLICATIONS

EstablishingEstablishing a a productionproduction processprocess forfor microalgaemicroalgaeas as sourcesource ofof bioethanolbioethanol’’ss feedstockfeedstockFactorsFactors toto be be consideredconsidered ((andand optimizedoptimized))

•• OrganismOrganism- natural isolate (production site) - strain from culture collection- carbohydrate overproducing mutant (?)

•• Culture systemCulture system- open, closed, semi?

•• Operating conditionsOperating conditions- batch, semi-continuous, continuous?- nutrient limitation(s)? - one-stage, two-stage?

A plausible (although ambitious) objective, A plausible (although ambitious) objective, considering present state of art (high considering present state of art (high

insolationinsolation area)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 m35 g biomass m--22 dayday--11

• For a carbohydrate content of ~30% = 10.5 g 10.5 g carbohydratecarbohydrate mm--22 dayday--11

• Surface extrapolation = 0.35 ton biomass (0.105 ton 0.35 ton biomass (0.105 ton carbohydrate) hacarbohydrate) ha--11 dayday--11

• Surface + time extrapolation (effective operation 300 days per annum) =105 ton biomass (31.5 ton carbohydrate) ha105 ton biomass (31.5 ton carbohydrate) ha--11

yearyear--1 1 ~~((19,000 L ethanol19,000 L ethanol) ha) ha--11 yearyear--11

MicroalgalMicroalgal metabolic pathways that can be leveraged for metabolic pathways that can be leveraged for biofuelbiofuel productionproduction

RadakovitsRadakovits et al. (2010) Eukaryotic Cell 9: 486et al. (2010) Eukaryotic Cell 9: 486--501501

Starch metabolism in green Starch metabolism in green microalgaemicroalgae

RadakovitsRadakovits et al. (2010) Eukaryotic Cell 9:486et al. (2010) Eukaryotic Cell 9:486--501501

FermentativeFermentative productionproduction ofof bioethanolbioethanolRaw materialsRaw materials

• Sugar cane (Brazil)

• Corn (USA)

• Wheat, corn, sugar beet (Europe)

• Alternatives: lignocellulosic materials; microalgae

Alcoholic fermentation (yeasts)

C6H12O6 2 CH3CH2OH + 2 CO2

COCO22 emissionsemissions

(16 kJ g-1) (30 kJ g-1)

Ethanol Ethanol photoproductionphotoproduction from COfrom CO22

2 CO2 CO22 + 3 H+ 3 H22O O →→ CHCH33CHCH22OH + 3 OOH + 3 O22

COCO2 2 fixationfixation EthanolEthanol photosynthesisphotosynthesis

LIGHT

PDCACETALDEHYDE ETHANOL

ADHPYRUVATEPYRUVATE

3-PGA

CO2CalvinCalvincyclecycle

SynechocystisSynechocystis spsp. PCC6803. PCC6803((SectionnSectionn I , I , RippkaRippka et al.et al., 1979), 1979)

Model Model cyanobacteriumcyanobacteriumGrowth on glucoseGrowth on glucoseFull genomic sequence available Full genomic sequence available ((http://http://www.kazusa.or.jpwww.kazusa.or.jp))Transformable (chromosome and Transformable (chromosome and plamidplamid))Homologous recombinationHomologous recombination

•• Fast growthFast growth•• Easy cultureEasy culture

1. Insertion in 1. Insertion in SynechocystisSynechocystis genome of genome of ZymomonasZymomonas genes involved in ethanol genes involved in ethanol synthesis through homologous recombinationsynthesis through homologous recombination

Secuence homologous toSynechocystis DNA (needed for reombination)

Endogenous promotor (externally inducible)

Pyruvate decarboxylase and alcohol dehydrogenase genes

Antibiotic-resistance cassette

StrategyStrategy forfor obtainingobtaining SynechocystisSynechocystis strainsstrains ableable toto synthesizesynthesizeethanolethanol

pdc-adhPP

PP

2. 2. AnalysisAnalysis ofof properproper integrationintegration in in genomegenome, , andand ofof full full segregationsegregation, by Southern , by Southern BlotBlot

3. 3. ExpressionExpression analysisanalysis ofof genes in a single genes in a single RNAmRNAm underunder inducinginducing conditionsconditions, by , by NorthernNorthern BlotBlot

4. 4. MeasurementMeasurement ofof enzymeenzyme activitiesactivities in in cellcell extractsextracts underunder inducinginducing conditionsconditions

5. 5. VerificationVerification ofof ethanolethanol presencepresence in in outerouter mediummedium