Butanol: A Second Generation Biofuel

Hans P. Hans P. BlaschekBlaschekUniversity of IllinoisUniversity of Illinois

April 14, 2006April 14, 2006

Outline

IntroductionIntroductionHistoryHistoryMetabolic PathwayMetabolic Pathway

C. C. beijerinckiibeijerinckii Strain Development and Strain Development and CharacterizationCharacterization

PostPost--genomic Characterizationgenomic CharacterizationDownstream Processing and RecoveryDownstream Processing and Recovery

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

Production of Butanol from Corn

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

Commercial Solvents Corp.Terra Haute, Indiana

Rationale

ButanolButanol is an important industrial chemical is an important industrial chemical and feedstockand feedstockSuperior fuel extenderSuperior fuel extenderFoodgradeFoodgrade extractantextractant

Advantages as Liquid Fuel:

Higher energy content than ethanolHigher energy content than ethanolCan be stored under humid conditions Can be stored under humid conditions unlike ethanolunlike ethanol

lack of solubility with waterlack of solubility with waterCan be used in internal combustion and Can be used in internal combustion and diesel enginesdiesel enginesCan be transported by pipelineCan be transported by pipeline

CharacteristicCharacteristic GasolineGasoline DieselDiesel MethanolMethanol EthanolEthanol ButanolButanol

FormulaFormula C4C4--C12 C12 hydrocarbonshydrocarbons

C14C14--C20 C20 hydrocarbonshydrocarbons

CHCH33OHOH CHCH33CHCH22OHOH CHCH33(CH(CH22))33OHOH

Boiling PointBoiling Point

ooCC 3232--210210 204204--343343 6565 7878 118118

ooFF 9090--410410 400400--650650 149149 173173 244244

Lower heating Lower heating value*value*

MJ/kgMJ/kg 44.544.5 43.043.0 19.619.6 26.926.9 33.133.1

Btu/galBtu/gal 114,800114,800 140,000140,000 55,61055,610 76,10076,100 96,10096,100

Liquid Fuel Characteristics

Butanol MarketWorldwide butanol market 4.5 x 10Worldwide butanol market 4.5 x 109 9 lb/year. Of lb/year. Of this approximately one half is produced in the this approximately one half is produced in the United StatesUnited StatesThis would translate into the utilization of 400 This would translate into the utilization of 400 million additional bushels of corn or equivalent million additional bushels of corn or equivalent amount of agricultural biomass annuallyamount of agricultural biomass annually2005 average price: $0.70/lb2005 average price: $0.70/lb3% Annual increase in demand3% Annual increase in demand

Currently......

BioBio--ButanolButanol produced in China produced in China Small niche market in the food and flavor Small niche market in the food and flavor industries for fermentationindustries for fermentation--derived derived butanolbutanolThe finite nature and instability of fossil The finite nature and instability of fossil fuels will ultimately lead to rising fuels will ultimately lead to rising petrochemical pricespetrochemical prices

ABE Fermentation Products

ButanolButanol (6)(6)Acetone (3)Acetone (3)Ethanol (1)Ethanol (1)

chemical derivatives:chemical derivatives:Butyl and Butyl and methacrylatemethacrylate estersesters1,31,3--Butadiene (synthetic rubber)Butadiene (synthetic rubber)butyl acetate (solvent for paint lacquers)butyl acetate (solvent for paint lacquers)

Ethanol

Glucose

PyruvateLactate

Acetyl-CoAAcetyl-PAcetate Acetylaldehyde

Acetoacetyl-CoAAcetoacetateAcetone

Butyryl-CoAButyryl-PButyrate Butyraldehyde

ButanolPTBBK BADH BDH

AK PTA ADH EDH

CoATAADC

ABE Metabolic Pathway: Biphasic Fermentation

Clostridium beijerinckii BA101

BA101 strain developed in the early 1990BA101 strain developed in the early 1990’’s using s using chemical mutagenesis together with selective chemical mutagenesis together with selective enrichment on the nonenrichment on the non--metabolizablemetabolizable glucose glucose analog, 2analog, 2--deoxydeoxy--glucoseglucosePilot Scale (20Pilot Scale (20--liter) Fermentations using 6% liter) Fermentations using 6% glucose or glucose or maltodextrinmaltodextrin demonstrated that BA101 demonstrated that BA101 produces produces twicetwice as much as much butanolbutanol as parent strain as parent strain In addition, BA101 exhibits reduced acid In addition, BA101 exhibits reduced acid production and increased carbohydrate utilizationproduction and increased carbohydrate utilization

C. beijerinckii strain comparison

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20

Butanol Concentration [g/L]

But

anol

Pro

duct

ivity

[g/L

.h]

8052

BA101

C. beijerinckiiC. beijerinckii BA101 demonstrated higher BA101 demonstrated higher productivity at a given productivity at a given butanolbutanol concentrationconcentration

C. beijerinckii BA101: Characteristics

C. C. beijerinckiibeijerinckii BA101* is able to BA101* is able to produce up to 20g produce up to 20g butanol/lbutanol/l and 33 and 33 g/L total solventsg/L total solvents

Hyper Hyper amylolyticamylolyticHyper Hyper butanolbutanol producingproducingLow acid producingLow acid producingStableStable

––*U.S. Patent 6,358,717*U.S. Patent 6,358,717

Modeling Studies

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

N. N. QureshiQureshi and H.P. and H.P. BlaschekBlaschek. 2001. ABE production . 2001. ABE production from corn: A recent economic evaluation. J. from corn: A recent economic evaluation. J. Industrial Industrial MicrobiolMicrobiol. & . & BiotechnolBiotechnol. 27:292. 27:292--297297..

Enhanced solvent production by C. beijerinckii BA101:

Several key enzymes in solvent formation Several key enzymes in solvent formation pathway have elevated levels of activitypathway have elevated levels of activitye.g. e.g. butyraldehydebutyraldehyde dehydrogenasedehydrogenaseCoACoA transferasetransferasephosphotransbutyrylasephosphotransbutyrylase activitiesactivities

The Glucose Utilization by C. beijerinckii BA101 and NCIMB 8052.During 1L batch fermentation, cultures were grown in 6% glucose P2medium at 33oC with 5% inoculum.

Time (Hour)

0 10 20 30 40 50

OD

600

0

1

2

3

4

5

6

Glu

cose

(g/L

)

10

20

30

40

50

60

The growth of C.beijerinckii BA101 The growth of C. beijerinckii NCIMB 8052 Glucose Utilization by C.bejerinckii BA101 glucose utilization by C. beijerinckii NCIMB 8052

Examination of Carbohydrate uptake and transfer in C. beijerinckii BA101

ATP-dependent sugar transport

The presence of a highly active alternative The presence of a highly active alternative transport mechanism may be the reason for transport mechanism may be the reason for why why C. C. beijerinckiibeijerinckii BA101 is capable of BA101 is capable of better glucose utilization during better glucose utilization during fermentation than the wild type strain in fermentation than the wild type strain in spite of a defect in PTS.spite of a defect in PTS.

–– Lee et al. Lee et al. ApplAppl. Environ. Microbiol.71:3384. Environ. Microbiol.71:3384--3387 (2005)3387 (2005)

Sequencing Sequencing C. C. beijerinckiibeijerinckiiNCIMB 8052NCIMB 8052

C. C. beijerinckiibeijerinckii NCIMB 8052 genome NCIMB 8052 genome sequenced by JGIsequenced by JGI--DOEDOE

Los Alamos National Lab genome finishing team Los Alamos National Lab genome finishing team --closing closing 5.9 5.9 mbmb in 89 in 89 contigscontigs of 20 reads or greater, of 20 reads or greater, 29.8 % GC; 4117 candidate protein29.8 % GC; 4117 candidate protein--encoding gene encoding gene modelsmodelsCommunity Annotation Process underway using Community Annotation Process underway using Manatee web based systemManatee web based system

http://genome.jgihttp://genome.jgi--psf.org/draft_microbes/clobe/clobe.home.htmlpsf.org/draft_microbes/clobe/clobe.home.html

Post-Genomic Analysis

Post transcriptional analysis of global shift in gene Post transcriptional analysis of global shift in gene expression during transition from expression during transition from acidogenesisacidogenesis to to solventogenesissolventogenesisExamination of differences in gene expression Examination of differences in gene expression between hyperbetween hyper--butanolbutanol producing producing C. C. beijerinckiibeijerinckiiBA101 and wild type 8052 strainsBA101 and wild type 8052 strainsComparison with Comparison with C. C. acetobutylciumacetobutylcium ATCC 824 ATCC 824 genome sequencegenome sequence

Limitation: Butanol Toxicity

Batch cultures are selfBatch cultures are self--limiting due to limiting due to toxicity of butanol to producing culture toxicity of butanol to producing culture Toxicity results in low concentrations of Toxicity results in low concentrations of butanol in fermentation brothbutanol in fermentation brothHigh energy inputs required to recover High energy inputs required to recover butanol from dilute streamsbutanol from dilute streams

How do we overcome this?How do we overcome this?

ABE Process Development

Culture development and characterizationCulture development and characterizationFermentation optimizationFermentation optimizationProduct recoveryProduct recoveryProcess integrationProcess integrationUse of less expensive substratesUse of less expensive substrates

Alternative SubstratesAlternative Substrates

StarchStarch--based Packaging Peanuts based Packaging Peanuts ((EzejiEzeji et al., 2003)et al., 2003)

Agricultural Waste Streams (Jesse et Agricultural Waste Streams (Jesse et al., 2002)al., 2002)

DeDe--germedgermed Corn (Campos et al., 2002)Corn (Campos et al., 2002)

Requires supplementation with CSLRequires supplementation with CSL

Soy Molasses (Soy Molasses (QureshiQureshi et al., 2001)et al., 2001)

Corn Fiber (Corn Fiber (EzejiEzeji and and BlaschekBlaschek, , 2005)2005)

Distillers Grains (Distillers Grains (EzejiEzeji and and BlaschekBlaschek, 2005), 2005)

ABE Fermentation by ABE Fermentation by Clostridium Clostridium beijerinckiibeijerinckiiusing using xylosexylose or glucose (30 g/L) as or glucose (30 g/L) as

substratesubstrate

0

2

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6

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10

12

14

0 24 36 48 60 720

0.5

1

1.5

2

2.5

XyloseGlucoseXylose Cell Conc.(g/L)Glucose Cell Conc.(g/L)

ABE Conc. (g/l)

CellConc.(g/l)

Fermentation of Fermentation of HexoseHexose and Pentoseand Pentosesugars for ABE production by sugars for ABE production by solventogenicsolventogenic clostridiaclostridia

0

5

10

15

20

25

Products [g/L]

Total ABE Butanol Acetone Ethanol

C. acetobutylicum 260C. acetobutylicum 824C. saccharobutylicum 262C. butylicum NRRL 592C. beijerinckii 8052C. beijerinckii BA101

Glucose-mannose-arabinose-xylose composite (g/L: 25:5:10:20)

Fermentation of mixed Fermentation of mixed hexosehexose and and pentose sugars by pentose sugars by C. C. beijerinckiibeijerinckii

BA101BA101ParametersParameters **Control**Control *GMAX*GMAX

Acetone (Acetone (g/lg/l)) 4.84.8 3.53.5

Ethanol (Ethanol (g/lg/l)) 0.450.45 0.570.57

ButanolButanol ((g/lg/l)) 13.013.0 13.913.9

Total ABE (Total ABE (g/lg/l)) 18.218.2 17.917.9

Total Acids (Total Acids (g/lg/l)) 0.870.87 0.720.72

Fermentation time Fermentation time (h)(h)

6060 7272

Productivity Productivity ((g/l/hrg/l/hr))

0.300.30 0.250.25

Utilization of Mixed Utilization of Mixed SugarsSugars

Various Various solventogenicsolventogenic clostridia utilized a mixed clostridia utilized a mixed sugar stream for ABE production. sugar stream for ABE production.

Although glucose is the preferred carbon source, Although glucose is the preferred carbon source, these microbes have the ability to concurrently these microbes have the ability to concurrently utilize different (utilize different (hexosehexose and pentose) sugars for and pentose) sugars for ABE productionABE production..

05

10152025

0 20 40 60 80 100Fermentation Time [h]

Res

idua

l sug

ars

[g/L

]

05

1015

2025

0 20 40 60 80 100Fermentation Time [h]

Res

idua

l sug

ars

[g/L

]

C. beijerinckii BA101 C. acetobutylicum 824

Integrated Fermentation Process• Eliminate product inhibition (butanol toxicity)

• Achieve high productivity

• Produce concentrated products (acetone-butanol)

• Utilize concentrated substrates - to allow for a reduction in process volume

Gas Stripping

No expensive apparatusNo expensive apparatusDoes not foulDoes not foulDoes not remove nutrients or intermediates Does not remove nutrients or intermediates (e.g. acids)(e.g. acids)Useful for reducing Useful for reducing butanolbutanol concentration concentration below physiologicallybelow physiologically--toxic levelstoxic levelsAllows for recovery of concentrated AB Allows for recovery of concentrated AB product streamsproduct streams

Gas stripping: Integrated Gas stripping: Integrated process for production and process for production and

recoveryrecovery

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

Integrated vs. NonIntegrated vs. Non--Integrated Integrated Batch (1L) FermentationsBatch (1L) Fermentations

Parameters Parameters (g/l)(g/l)

NonNon--IntegratedIntegrated

Integrated Integrated BatchBatch

Integrated Integrated Fed Batch*Fed Batch*

AcetoneAcetone 5.35.3 6.96.9 27.427.4

ButanolButanol 11.911.9 16.416.4 46.446.4

EthanolEthanol 0.50.5 0.30.3 2.12.1

Acetic acidAcetic acid 0.50.5 00 00

Butyric acidButyric acid 0.40.4 00 00

Total ABETotal ABE 17.717.7 23.623.6 75.975.9

Total acidsTotal acids 0.90.9 00 00

CellsCells 3.23.2 4.64.6 1111

Initial Initial GlucoseGlucose

59.259.2 59.759.7 161.7161.7

Residual Residual GlucoseGlucose

14.614.6 00 00

Glucose used Glucose used (%)(%)

75.375.3 100100 100100

ABE Prod ABE Prod g/l/hg/l/h

0.290.29 0.610.61 0.600.60

ABE YieldABE Yield 0.40.4 0.40.4 0.470.47

Integrated processes employing Gas stripping

Fermentation and gas stripping were integrated in batch, Fermentation and gas stripping were integrated in batch, fedfed--batch and continuous systems:batch and continuous systems:

-- In a batch reactor/gas stripping processIn a batch reactor/gas stripping process75.975.9 g/L ABE was produced g/L ABE was produced

-- In a fedIn a fed--batch reactor/gas strippingbatch reactor/gas strippingprocess process 232232 g/L ABE was producedg/L ABE was producedand and 500500 g/L glucose was utilized g/L glucose was utilized

-- In a continuous system/gas strippingIn a continuous system/gas strippingprocess process 460460 g/L ABE was produced and g/L ABE was produced and 11631163 g glucose was utilized g glucose was utilized

SummarySummary

Integrated gas stripping Integrated gas stripping combined with fed batch combined with fed batch fermentation employing fermentation employing C. C. beijerinckiibeijerinckii BA101 demonstrates BA101 demonstrates potential for scale up.potential for scale up.

Genomic sequence information Genomic sequence information will allow for postwill allow for post--transcriptional analysis of transcriptional analysis of C. C. beijerinckiibeijerinckii 8052 and BA101.8052 and BA101.

Future WorkFuture Work

ScaleScale--up the integrated up the integrated butanolbutanolfermentation using fermentation using Clostridium Clostridium beijerinckiibeijerinckii BA101 and recovery by BA101 and recovery by gas strippinggas stripping

Examination of Corn Fiber Examination of Corn Fiber hydrolysateshydrolysates as substrate for as substrate for growth and ABE productiongrowth and ABE production

Developing a second generation Developing a second generation strain of strain of C. beijerinckiiC. beijerinckii BA101BA101

AcknowledgementsAcknowledgements

United States Department of United States Department of AgricultureAgriculture

Illinois Missouri Biotechnology Illinois Missouri Biotechnology AllianceAlliance

Illinois Corn Marketing BoardIllinois Corn Marketing Board

Illinois Council on Food & Illinois Council on Food & Agricultural Research (CFAR)Agricultural Research (CFAR)

Department of EnergyDepartment of Energy