Bioethanol from microalgae
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Transcript of Bioethanol from microalgae
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