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Butanol from Lignocellulose
Prof. Dr. Eckhard BolesInstitute of Molecular BiosciencesGoethe-University Frankfurt/Mainhttp://www.bio.uni-frankfurt.de/[email protected]
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: [email protected]
Thanks to: