ButanolButanol: A Second : A Second Generation Generation BiofuelBiofuel

Hans P. Hans P. BlaschekBlaschekUniversity of IllinoisUniversity of Illinois

March 6, 2007March 6, 2007

OutlineOutlineIntroductionIntroduction

HistoryHistoryRationaleRationale

Microbe Development and CharacterizationMicrobe Development and CharacterizationGenetic and PostGenetic and Post--genomic Characterizationgenomic CharacterizationSubstrate (CoSubstrate (Co--Product) UtilizationProduct) Utilization

Downstream Processing and RecoveryDownstream Processing and RecoveryIntegrated process Integrated process

ScaleScale--up and Commercialization of ABEup and Commercialization of ABE

Production of Production of ButanolButanol from from CornCorn

Commercial Plants were operational in Commercial Plants were operational in the USA to produce the USA to produce butanolbutanol from cornfrom corn

Commercial Solvents Corp.Commercial Solvents Corp.Terra Haute, IndianaTerra Haute, Indiana

Rationale

••ButanolButanol is an important industrial chemical is an important industrial chemical and feedstockand feedstock

••Superior fuel extender/replacementSuperior fuel extender/replacement

••FoodgradeFoodgrade extractantextractant

BioButanolBioButanol: A Second Generation : A Second Generation Liquid FuelLiquid Fuel

••Higher energy content than ethanolHigher energy content than ethanol

••Can be stored under humid conditions unlike ethanolCan be stored under humid conditions unlike ethanol--lack of solubility with water (higher flash point lack of solubility with water (higher flash point and lower vapor pressure)and lower vapor pressure)

••Can be used in internal combustion and diesel Can be used in internal combustion and diesel engines; less corrosiveengines; less corrosive

••Can be shipped through existing pipelinesCan be shipped through existing pipelines

••Replacement for gasoline or as a solventReplacement for gasoline or as a solvent

Characteristic Gasoline Diesel Methanol Ethanol Butanol

Formula C4-C12 hydrocarbons

C14-C20 hydrocarbons

CH3OH CH3CH2OH CH3(CH2)3OH

Boiling Point

oC 32-210 204-343 65 78 118

oF 90-410 400-650 149 173 244

Lower heating value*

MJ/kg 44.5 43.0 19.6 26.9 33.1

Btu/gal 114,800 140,000 55,610 76,100 96,100

Liquid Fuel Characteristics

C. beijerinckii BA101: CharacteristicsC. beijerinckii BA101* is able to produce up to 20g butanol/l and 33 g/L total solvents

Hyper amylolyticHyper butanol producingLow acid producingStable

*U.S. Patent 6,358,717

Solvent production using Solvent production using solventogenicsolventogenicClostridiaClostridia

Fermentation by C. beijerinckii NCIMB 8052 in laboratory batch culture typically yields butanol, acetone and ethanol in a 6:3:1 ratio.

BioBio--ButanolButanol: Fermentation : Fermentation strain comparisonstrain comparison

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Butanol Concentration [g/L]

But

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8052

BA101

Clostridial life cycleClostridialClostridial life cyclelife cycle

Solventogenesis/Acidogenesis

Aminosugar metabolism

Stress responses

Fatty acid biosynthesis

operon

Fatty acid metabolism

Glycolysis

Topology

Motility/ Chemotaxis

Transcriptional regulators

Sporulation

Genes profiled in C. beijerinckiimicroarray

Genomic analysis of Genomic analysis of C. C. beijerinckiibeijerinckii NCIMB NCIMB 80528052

JGIJGI--DOE sequenced the DOE sequenced the C. C. beijerinckiibeijerinckii NCIMB 8052 genomeNCIMB 8052 genomeDetermination of Gene FunctionsDetermination of Gene Functions

www.jgi.doe.gov/JGI_microbial/htmlwww.jgi.doe.gov/JGI_microbial/html//

PostPost--Genomic AnalysisGenomic Analysis

Post transcriptional analysis of global shift Post transcriptional analysis of global shift in gene expression during transition from in gene expression during transition from acidogenesisacidogenesis to to solventogenesissolventogenesisExamination of differences in gene Examination of differences in gene expression between hyperexpression between hyper--butanolbutanolproducing producing C. C. beijerinckiibeijerinckii BA101 and wild BA101 and wild type 8052 straintype 8052 strainRoadmap for development of next Roadmap for development of next generation fermentation microorganismsgeneration fermentation microorganisms

Clostridial pathway reactions for solvent productionClostridialClostridial pathway reactions for solvent productionpathway reactions for solvent production

butyryl-CoAdehydrogenase

Pyruvate ferredoxinoxidoreductase

Acetyl-CoA

Pyruvate

Acetoacetyl-CoA

3-Hydroxybutanoyl-CoA

Crotonyl-CoA

Butyryl-CoA

thiolase

crotonase

3-hydroxybutyryl-CoA dehydrogenase

Butanol

Ethanol

Acetone

Hexose

alcohol dehydrogenase

butanoldehydrogenase II

Butyrylphosphate

Butyrate

Acetylphosphate

Acetate

aceto-acetyl CoA: acetate-butyrate CoA transferasesubunit a/b

acetoacetatedecarboxylase

Glycolysis

phosphate butyryltransferase

butyrate kinase

phosphotransacetylaseacetate kinase

(Embden Meyerhof pathway)

Transcriptional analysis of Transcriptional analysis of C. C. beijerinckiibeijerinckii

Quantification of Quantification of gene expression gene expression on arrayson arrays

Reverse Reverse transcriptiontranscription

Dye Dye labellinglabelling

RNA RNA extractionextraction

ClostridialClostridialcultureculture

cDNAscDNAs LabelledLabelled probesprobesClostridialClostridial RNAsRNAs

Long Long oligosoligos Printed arraysPrinted arrays

HybridizationHybridizationImmobilization Immobilization on glass slideson glass slides

Fluorescence Fluorescence image image scanningscanning

Genomic Genomic informationinformation Hybridized slidesHybridized slides

WildWild--typetype BA101BA101

Growth Response of Growth Response of C. C. beijerinckiibeijerinckii 80528052 and and hyperhyper--butanolbutanol producing BA101 mutantproducing BA101 mutant

acidogenic solventogenic

BA101Wild-type

Solventogenic/acidogenic genes – butanolproduction

Solventogenic/acidogenicSolventogenic/acidogenic genes genes –– butanolbutanolproductionproduction

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Expr

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NADHNADH--dependent dependent butanolbutanoldehydrogenasedehydrogenase II (BDHII)II (BDHII)

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Alcohol Alcohol dehydrogenasedehydrogenase

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Alcohol Alcohol dehydrogenasedehydrogenase

Expression of alcohol Expression of alcohol dehydrogenasesdehydrogenases: :

•• Increase by ~10 fold to a maximal level Increase by ~10 fold to a maximal level during during acidogenicacidogenic phase in wildphase in wild--type and type and BA101. BA101.

•• Expression is at maximal level in BA101 Expression is at maximal level in BA101 during during solventogenicsolventogenic phase, but downphase, but down--regulated to basal level in wildregulated to basal level in wild--type.type.

BA101BA101WildWild--typetype

Solventogenic/acidogenic genes – ptb-butK operon

Solventogenic/acidogenicSolventogenic/acidogenic genes genes –– ptbptb--butKbutK operonoperon

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Butyrate Butyrate kinasekinase ((bkbk))

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Acetate Acetate kinasekinase ((akak))

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Expr

essi

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•• Expression of Expression of bkbk, , akak and PTA is similar during and PTA is similar during acidogenicacidogenic phase for both wildphase for both wild--type and BA101, type and BA101, while expression level is higher for BA101 by up while expression level is higher for BA101 by up to 10 fold during to 10 fold during solventogenicsolventogenic phase vs. wildphase vs. wild--type. type.

•• Expression remains constant in BA101, but Expression remains constant in BA101, but decreases by ~10 fold in decreases by ~10 fold in solventogenicsolventogenic phase phase for wildfor wild--type. type.

•• AkAk and PTA expression is lower during and PTA expression is lower during solventogenesissolventogenesis, although less pronounced in , although less pronounced in BA101 than in wildBA101 than in wild--type. type.

BA101BA101WildWild--typetype

Gene expression for terminal enzymes in solvent Gene expression for terminal enzymes in solvent formation pathwayformation pathway

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adc0.1

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ctfA0.01

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ctfB

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adh0.1

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bdhIIMutantWild-type

Gene expression for pyruvate conversion to solventsGene expression for Gene expression for pyruvatepyruvate conversion to solventsconversion to solvents

3-hydroxybutyryl-CoA dehydrogenase

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thiolase

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pyruvate ferredoxin oxidoreductase

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crotonase

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butyryl-CoA dehydrogenase butyrate kinase

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Acetate kinase

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Phosphotransacetylase

MutantWild-type

8052 Wild-type

BA101 Mutant

Temporal profiling of gene expression during the ABE fermentation by C. beijerinckii

Temporal profiling of gene expression during the ABE Temporal profiling of gene expression during the ABE fermentation by fermentation by C. C. beijerinckiibeijerinckii

Changes in global gene expression appear Changes in global gene expression appear more robust in 8052 wildmore robust in 8052 wild--type cells type cells than in the BA101 mutant. than in the BA101 mutant.

Gene activation, mostly in Gene activation, mostly in sporulationsporulation during during the the solventogenicsolventogenic phase is prominent in phase is prominent in the wildthe wild--type, but less distinct in type, but less distinct in mutant. mutant.

Repression of genes, many in Repression of genes, many in glycolysisglycolysis, , during during solventogenicsolventogenic phase is phase is pronounced in wildpronounced in wild--type, less so in type, less so in mutant. mutant.

Genomic organization of terminal enzymes in the Genomic organization of terminal enzymes in the solventogenicsolventogenic pathwaypathway

Acetone production enzymes ctfA/ctfB and adc are co-located in a single operon in the C. beijerinckii genome, consistent with their clustering in expression analysis.

Biological ConversionBiological ConversionFermentation Fermentation -- utilization of 5 & 6 carbon sugarsutilization of 5 & 6 carbon sugarsExamples: Examples:

ZymomonasZymomonas mobilismobilis engineered to ferment engineered to ferment xylosexylose and and arabinosearabinose, , S. S. cerevisiaecerevisiae strains engineered to ferment strains engineered to ferment arabinosearabinose,,SolventogenicSolventogenic clostridia with abilityclostridia with abilityto simultaneously to simultaneously saccharifysaccharify and ferment.and ferment.

Turning Corn Stover into Turning Corn Stover into ValueValue--Added ProductsAdded Products

Could be collected as used as a Could be collected as used as a substratesubstrate

Current Treatment Technologies:Current Treatment Technologies:Pretreatment with steam and acid followed by Pretreatment with steam and acid followed by enzymatic hydrolysis to produceenzymatic hydrolysis to produce90 M tons of fermentable sugars available for90 M tons of fermentable sugars available forconversion to liquid fuels & chemicalsconversion to liquid fuels & chemicals

Substrates/CoSubstrates/Co--ProductsProductsDDGS and Corn Fiber (DOE/Midwest DDGS and Corn Fiber (DOE/Midwest Consortium)Consortium)StarchStarch--based Packaging Peanuts (based Packaging Peanuts (EzejiEzeji et al., et al., 2003)2003)Agricultural Waste Streams (Jesse et al., Agricultural Waste Streams (Jesse et al., 2002)2002)DeDe--germedgermed Corn (Campos et al., 2002)Corn (Campos et al., 2002)Soy Molasses (Soy Molasses (QureshiQureshi et al., 2001)et al., 2001)

Integrated ProcessIntegrated Process•• Eliminate product inhibition (butanol toxicity)Eliminate product inhibition (butanol toxicity)•• Achieve high productivityAchieve high productivity•• Produce concentrated products (acetoneProduce concentrated products (acetone--

butanol)butanol)•• Utilize concentrated substrates Utilize concentrated substrates -- to allow for to allow for

a reduction in process volumea reduction in process volume•• Better economicsBetter economics

Gas stripping: Integrated process for production and recovery

ABE

Condenser

Bioreactor

Gas recycle

Gas is bubbled through the fermentation broth and then circulated though the condenser. ABE captured in reactor is condensed in condenser and gas is recycled back to the reactor. ABE is recovered as liquid.

2 Liters

vessel

condenser

cooler

Circulatingpump

Modeling StudiesModeling StudiesEconomic modeling studies indicated that the Economic modeling studies indicated that the use of use of C. C. beijerinckiibeijerinckii BA101 hyperBA101 hyper--butanolbutanolproducing strain when used in combination producing strain when used in combination with improved recovery technologies can be with improved recovery technologies can be competitive to the petrochemical route for competitive to the petrochemical route for producing producing butanolbutanol..

N. N. QureshiQureshi and H.P. and H.P. BlaschekBlaschek. 2001. ABE production from corn: A recent . 2001. ABE production from corn: A recent economic evaluation. J. Industrial economic evaluation. J. Industrial MicrobiolMicrobiol. & . & BiotechnolBiotechnol. 27:292. 27:292--297.297.

SummarySummaryGenes encoding terminal Genes encoding terminal solventogenicsolventogenic enzymes exhibit rapid and large fold enzymes exhibit rapid and large fold increases in message levels (up to 100increases in message levels (up to 100--fold) during early exponential phase fold) during early exponential phase growth and the high expression level is sustained throughout growth and the high expression level is sustained throughout solventogenicsolventogenicphase. phase.

In the In the C. C. beijerinckiibeijerinckii BA101 hyperBA101 hyper--butanolbutanol producing mutant, expression of producing mutant, expression of a large number of genes including alcohol a large number of genes including alcohol dehydrogenasesdehydrogenases is maintained is maintained throughout the throughout the solventogenicsolventogenic stage, while these genes are repressed during stage, while these genes are repressed during the the solventogenicsolventogenic phase in the 8052 wildphase in the 8052 wild--type culture. type culture.

Repression of various Repression of various sporulationsporulation genes is stronger in the BA101 mutant genes is stronger in the BA101 mutant than in wildthan in wild--type strain. type strain.

Potential exists for utilization of 5 and 6 carbon sugars obtainPotential exists for utilization of 5 and 6 carbon sugars obtained from ed from lignocellulosiclignocellulosic--based based hydrolysateshydrolysates

Integrated approach for bioIntegrated approach for bio--butanolbutanol production and simultaneous product production and simultaneous product removal is promising and is currently being scaled up.removal is promising and is currently being scaled up.