Butanol from Lignocellulose

Prof. Dr. Eckhard BolesInstitute of Molecular BiosciencesGoethe-University Frankfurt/Mainhttp://www.bio.uni-frankfurt.de/bolese.boles@bio.uni-frankfurt.de

New Biofuels 20152nd/3rd March, Berlin

Biofuels from microorganisms

Rohstoff

CO2 +

Sonnenlicht pflanzliche Biomasse

Lignozellulose Stärke, freie Zucker

Syngas (CO + H2) C5 - Zucker C6 - Zucker

Algen Cyanobak-

terien

Synechocystis sp.

Synechococcus sp.

Bakterien

Clostridium sp.

Escherichia coli

Corynebact. glutamicum

Hefen

Saccharomyces cerevisiae

Pichia stipitis

Kluyveromyces sp.

Lipide, Isoprenoid-Derivate Ethanol, Butanol, Isobutanol und höhere Alkohole

Diesel Benzin

Produkte

Mikroorganismen

Acetogene

Bakterien

• higher energy content (95% versus 66%)

• lower vapour pressure

• higher flashpoint

• less miscible with water

• far less corrosive

• can be distributed through existing infrastructure

• can replace fossil fuels up to 100%

• can be blended with diesel

Biobutanol versus bioethanol

Butanols – family of 4-carbon alcohols (C4H9OH)

OH

OH

OH

OH

n-butanol (1-butanol)

isobutanol (2-methyl-1-propanol)

sec-butanol (2-butanol)

tert-butanol (2-methyl-2-propanol)

SolventsPlasticizersBuilding blocksCosmeticsFuel (additive)

n-butanol and isobutanol:

sec-butanol:

tert-butanol:

Applications of butanols

to butanone (methyl ethyl ketone MEK)(solvent)

gasoline antiknock additive

property n-butanol isobutanol sec-butanol tert-butanol

% heating value ofgasoline

84 83 83

water solubility(g/100mL)

7.7 8.0 12.5 miscible

motor octane number 81 90 93

liquid liquid liquid solid at roomtemperature

Properties of butanols

n-butanol: is the major natural product of ABE fermentations with bacteria(known technology, low yields = 15-20 g/L)

isobutanol: is produced in yeast fermentations as fusel alcohol (low amounts).some genetically modified microbial cells can produce it in higher amounts

sec-butanol: recently produced with genetically modified yeasts,normally produced by fermentation to 2,3-butanediol and chemical conversion

tert-butanol: not produced by any known biological route

Bio-butanol

Biobutanol producing microorganisms:

Solventogenic ClostridiaBakers yeastRecombinant bacteria

Second generation biofuels…… from lignocellulosic biomass

• High CO2 reduction potential (>90%)

• cheap

• No competition with food and feed production

• High energy yields from plant waste biomass and energy plants

• Necessary for diversification of transportation fuels,higher security of energy supplies

Spruce hydrolysate(stream and diluted sulfuric acid pretreatment)

– SEKAB E-Technology

Inhibitory substances:

~ 20 g/l organic acids(acetic acid, levulinic acid, formic acid)

~ 10 g/l aldehydes (Furfural, HMF)

phenolic compounds

Wheat straw hydrolysate (concentrated hydrochloric acid pretreatment) – Green Sugar GmbH

Inhibitory substances:

phenolic compoundshigh salt content

Maize silage hydrolysate (steam pretreatment)-University of Hohenheim

Inhibitory substances:

~ 50 g/l organic acids (acetic acid, lactic acid)

Lignocellulosic hydrolysates

Lignin 21-32%

Cellulose 33-51%

Extractives 1-5%

Hemicellulose 19-34%

sugars...

glucose

mannose

galactose

rhamnose

xylose

arabinose

HEXOSES

PENTOSES

O

OH

CH2OH

OH

OH

OH

OCH2OH

OH

OH

OHOH

OOH

OH

CH2OH

OHOH

OCH3

OH

OH OH

OH

O

OH

OH

OH

OH

O

OH

OH

OH

HOH2C

FERMENTATIONINHIBITORS

Lignocellulosic hydrolysates

320kg 190kg 24kg

Glucose� BuOH Xylose� BuOH Arabinose� BuOH

208 kg BuOH/ ton straw

Normal yeasts

95%125kg

95%74 kg

95%9 kg

yield:kg of BuOH:

kg sugar/ton straw

Conversion of C5/ C6-sugars to butanol

95%125kg

yield:kg of BuOH:

125 kg BuOH/ ton straw

C6/C5-yeasts

Conversion of 1 ton straw

Pentose fermentation enables increased product yields fromlignocellulosic biomass hydrolysates

Pentose fermentation

Solventogenic Clostridium species Saccharomyces cerevisiae yeasts

- Poor tolerance to inhibitors (detoxification required)

- Low yield of butanol

- Can ferment hexoses and pentoses

- Alcohol production coupled to growth- 2 stage fermentation (acidogenesis/solventogenesis)fermentation process difficult to control

- Bacteriophage infections/degeneration

- Strictly anaerobic conditions

Butanol recovery technologies

- Easy to be modified genetically

- Specific media requirements und cultivation conditions

- Byproducts

- Only trace amounts of isobutanol

- Poor tolerance to butanol

- Ethanol as the main product

- No pentose fermentation

Advantages of S. cerevisiae yeasts

Fermentation

• Yeasts are among the best known microorganisms and are easy to genetically modify

• Yeasts have GRAS (Generally Recognized as Safe) status

• Yeasts are well established in production processes with a high robustness

• Yeasts are highly resistant to toxic inhibitors and fermentation products, and ferment sugars at low pH values

• Yeast production processes have a minimized contamination risk

• All ethanol production facilities worldwide use yeasts and yeasts have a high acceptance among ethanol producers

• Recombinant yeast strains are available which can ferment pentoses and do no longer produce ethanol

Lesaffre/(Butalco/Uni Frankfurt)

DuPont/(Verdezyne)

DSM/(Royal Nedalco)

Lallemand/(Mascoma)

Clariant/(Süd-Chemie)

Terranol (with Novozymes)

C5/C6 yeasts ready for commercial use

Butalco is developing proprietary technologies to produce second generation biofuels and other biochemicals with yeast

Business model

• Start-up company of University of Frankfurt

• Development and commercialisation of yeast technology to produce 2nd generation biofuels and biochemicals

• Development of technologies in the field of lignocellulose hydrolysis, fermentation technology and downstream processing together with partners

• Build-up of a strong intellectual property position and licensing business

• Co-operation with producing companies but no own production facilities

• Founded in August 2007 with its head-quarters/commercial activities in Fuerigen / Switzerland

• Research activities with up to 5 scientists at the Universities of Frankfurt and Stuttgart-Hohenheim

• 2 own and 4 patents/patent applications bought from University of Frankfurt claiming important bottleneck technologies

• Wind energy company Volkswind as investor

• Summer 2012: Break-even sale of xylose isomerase technology to Lesaffre

Achievements

Realisation Strategy - Key Facts on Butalco

Butalco was sold to Lesaffre in the middle of 2014

Realisation Strategy - Exit Strategy

Exit strategy

Development of C5

technology

Development of biobutanol yeasts using

C5 and C6 sugars

(Yeasts producing other

biochemical)

Sale of the whole or

parts of the technology

portfolio

Sale of xylose technology

to Lesaffre

Out-licensing to

biofuel/biochemical producers

2011 20172013 20152012 2014 2016

Out-licensing to

biofuel/biochemical producers

Other activities

Aim of all shareholders always was an exit within the next 5 years

Sale of Butalco to Lesaffre

Glucose

Ethanol

Energie

Hefe

Sugar metabolism of yeast cells

Xylose

Glucose

Ethanol

Energie

Xylose

Engineering xylose fermentation

Clostridium

phytofermentans

Xylose Isomerase

D-Xylulose

D-Xylose

Pentose phosphate pathway

D-Xylulose-5-phosphate

spec. Xylose-Transporters

Kinase

Xylose isomerase from Clostridium phytofermentans

Vmax

(µmol min-1

mg protein-1)Km

(mM)

Ki (mM xylitol)

0.034 66 14.5

Xylose fermentation with S. cerevisiae

Farwick et al. (2014) PNAS 111, 5159

Brat et al. (2009) AEM 75, 2304

L-Ribulose-5-phosphate

L-Ribulose

Transporters

Pentose phosphate pathway

D-Xylulose-5-phosphate

4-Epimerase

Kinase

L-Arabinose

Becker and Boles Appl Environ Microbiol, 2003, 69:7, p. 4144Wiedemann and Boles Appl Environ Microbiol, 2008, 74:7, p. 2043Subtil and Boles Biotechnol Biofuels, 2011, 4:38

L-Arabinose pathway

L-arabinose transporter (AraT)from Pichia stipitis

L-arabinose isomerase (AraA)from Bacillus licheniformis

L-ribulokinase (AraB)from E.coli

L-ribulose-5-p 4-epimerase (AraD)from E.coli

Bacillus licheniformis

Arabinose Isomerase

Arabinose fermentation with S. cerevisiae

Yeast strain engineering for xylose fermentation

EthanolRed

0

10

20

30

con

cen

trat

ion

[g

/kg

]

HDY.GUF5 HDY.GUF6 HDY.GUF8

HDY.GUF9

0 10 20 300

10

20

30

time [h]

conc

entr

atio

n [

g/k

g]

HDY.GUF10

0 10 20 30time [h]

HDY.GUF11

0 10 20 30time [h]

HDY.GUF12

0 10 20 30

GlukoseXyloseArabinoseGlycerinEthanolBiomasse

time [h]

Mineral medium

C5 yeast CelluXTM 3 is commercially available

CelluX 3

Dry yeast for ethanolDeveloped and supplied by Leaf Technologies/Lesaffre

C5-technology developed by University of Frankfurt/Butalco

Glc

Gap

Pyr

Alac Dhiv Kiv

Iba

Isobutanol

Val

Ilv2 Ilv3

KAD

Ilv5Ilv6

EtOH

Bat1

ValBat2

Yeast produces isobutanol as a degradation product of valine metabolism

ADH

Cytosol

Mitochondrium

5 Glc 6 Xyl

Gap

Pyr KivIlv3

EtOH

ValBat2

Alac DhivIlv2 Ilv5

Iba

5 Isobutanol

Aro10

Pdc1

Pdc6

Pdc5

Alac Kiv Val

Ilv2Ilv3

Ilv5Ilv6

Bat1

Engineering isobutanol production in S. cerevisiae

ADH

NAD(P)HNAD(P)H

NAD(P)H

Pflbact.

Butalco technology

Proof-of-concept isobutanol fermentations

Brat et al. (2012) Biotechnol. Biofuels 5,65

∆ilv2

∆ilv5

∆ilv3

∆ilv2 ∆ilv5 ∆ilv3

WT

Optimized cytosolic valine pathway

0 10 20 30 400

10

20

30

40C

once

ntra

tion

[g/k

g]

0 10 20 30 400

1

2

3

Xyl

ose

Fer

men

tatio

nG

luco

seF

erm

enta

tion

Zeit [h]

0 50 100 150 2000

1

2

3

2,3-DihydroxyisovalerateAcetoin2,3-ButanediolIsobutanalIsobutanol

0 50 100 150 2000

10

20

30

40

XyloseEthanolGlycerolBiomass

Glucose Time (h)

Isobutanol fermentations with glucose and xylose

Butanol companies:

GevoButamaxGreenBiologicsRhodiaGranbioCobalt

FÖRDERKENNZEICHEN: 22031811 01.03.2012 bis 28.02.2015

Effiziente Co-Fermentation von Pentosen und Hexosen durch Hefen (ECO-FERM)

Johann Wolfgang Goethe-Universität Frankfurt am Main - FB 15 Biowissenschaften - Institut für Molekulare BiowissenschaftenMax-von-Laue-Str. 960438 Frankfurt am Main

Prof. Dr. Eckhard BolesTel: +49 69 798-29513E-Mail: e.boles@bio.uni-frankfurt.de

Thanks to: