Fermentative production of butanol – challenges and solutions
Peter Dürre Sao Paulo, July 25, 2012
Industrial use of butanol
bulk chemical precursor for production of
- acrylate and methacrylate esters
- glycol esters
- butyl acetate
- butylamines
- amino resins
used for production of adhesives/scalants, alkaloids, antibiotics, camphor, deicing fluid,
dental products, detergents, elastomers, electronics, emulsifiers, eye makeup, fibres
flocculants, flotation aids (e.g. butyl xanthate), hard-surface cleaners, hormones and
vitamins, hydraulic and brake fluids, industrial coatings, lipsticks, nail care products,
odorant standard, paints, paint thinners, perfumes, pesticides, plastics, printing ink,
resins, safety glass, shaving and personal hygiene products, surface coatings, super
absorbents, synthetic fruit flavoring, textiles, as mobile phases in paper and thin-layer
chromatography, as oiladditive, as well as for leather and paperfinishing
Advantages of butanol as a biofuel over ethanol
1. Can be blended in any concentration with gasoline (ethanol only
up to 85 %).
2. No modification of existing car engines required.
3. Has lower vapor pressure and is thus safer to handle.
4. Since it is not hygroscopic, blending is already possible in refinery.
5. Less corrosive. Complete existing infrastructure (tanks, pipelines,
pumps, filling stations, etc.) can be used.
6. Energy content is higher, resulting in a higher mileage/gasoline
blend ratio.
7. Dibutyl ether derivative has the potential for a diesel fuel.
Clostridium acetobutylicum -
biotechnological and political impact
(courtesy by H. Hippe)
-Isolation by Weizmann (between 1912 and 1914) in an research
project aiming at producing synthetic rubber from fermentation
products
-Large-scale fermentation of acetone for ammunition production
during World War I
-Balfour Declaration in 1917
-New production plants in Canada and the U.S.
-Stop of production after armistice in November 1918
-Introduction of prohibition in the U.S. in 1920, as a consequence
shortage of amyl alcohol, a solvent for laquers
-Henry Ford‘s assembly line automobile production required large
amounts of solvents for laquers
-Reopening of plants for butanol production (app. 2/3 of the world
market)
-After 1950, decline of the fermantation due to cheaper crude oil
prices
-Closure of last plants in South Africa (1982) and China (2004)
NCP, South Africa: 12 fermenters, working volume of 90,000 l each ( Jones, in: Clostridia (Bahl, Dürre, eds.))
Carbohydrates
Acid formation
Solvent formation Toxin synthesis
Clostridial form
Forespore
Spore
Spore maturation
Vegetative cells
Metabolism of Clostridium acetobutylicum
Hexose
Acetyl-CoA
Acetoacetyl-CoA
Butyryl-CoA Butyrate
Acetate Ethanol
Acetone
Adc
Butanol
AdhE
CtfA/B
Genes encoding solventogenic enzymes in
C. acetobutylicum
2000 4000 6000
orf5solBorfLadhEctfActfBadc
1000200030004000
adhE2 adcSadcR
500 1000150020002500
bdhB bdhA
megaplasmid
chromosome
Regulation of solvent formation
At the level of transcription:
Induction/repression controlled by known
transcription factors (Spo0A-P, CcpA, CodY)
as well as novel ones (AdcR/AdcS),
Posttranscriptional:
mRNA processing of the sol operon transcript
Posttranslational:
Protein modification of acetoacetate decarboxylase
Distribution of regulator binding sites
Genes encoding solventogenic enzymes are induced
by the master regulator Spo0A~ P
Ravagnani et al., Spo0A directly controls the swithch from acid to solvent production in
solvent-forming clostridia.
Mol. Microbiol. 37, 1172-1185, 2000
Most data stemming from investigation of C. beijerinckii.
Harris et al., Northern, morphological, and fermentation analysis of spo0A inactivation
and overexpression in Clostridium acetobutylicum ATCC824.
J. Bacteriol. 184, 3586-3597, 2002
Data stemming from investigation of C. acetobutylicum.
Problem solved, next question! Or?
acetone production after growth on MES medium
0
5
10
15
20
25
30
0 100 200 300 400
acet
on
e c
on
cen
trat
ion
[m
M]
time [h]
WT
DadcS 1
Dspo0A
wt
adcS73s::intron
spo0A462s::intron
0
10
20
30
40
50
60
70
80
90
0 100 200 300 400
bu
tan
ol c
on
cen
trat
ion
[m
M]
time [h]
butanol production after growth on MES medium
Growth of C. acetobutylicum WT and mutants on glucose
0
10
20
30
40
50
60
70
0 100 200 300 400
bu
tan
ol c
on
cen
trat
ion
[m
M]
time [h]
0
0,5
1
1,5
2
2,5
3
3,5
4
0 100 200 300 400
acet
on
e c
on
cen
trat
ion
[m
M]
time [h]
WT 1
DadcS 1
Dspo0A 2
wt
adcS73s::intro
n spo0A462s::intron
3.5
2.5
1.5
0.5
Growth of C. acetobutylicum WT and mutants on glucose
plus glycerol
acetone production after growth on MES medium butanol production after growth on MES medium
Regulator binding sites upstream of the sol promoter
Effect of solB overexpression on solventogenesis
200 nt –
Northern blot experiments
for verifying solB presence under
physiological conditions
Transcriptome of the sol operon region
solventogenic phase
acidogenic phase
A
T
A
A
T
A
A
A
G
T
C
T
T
C
A
G
A
T
G
T
T
T
A
A
T
T
C
C T A G
Primer extension experiments
for identification of solB transcription
start point
1 TAAGATATAG CTTCTTTTAT GTAGTATTAT TTCAGAAGTC TACAAATTAA GTTTATATTT
61 AGACCCTGGG GTGTAACTAT AGTATTTAAT ATTGGTACTA TTAATTAGGG TTATATATAC
121 TAGAACTTAT CATGGTAAAC ATAAATATAA ACTCAATTCT ATTTATGCTC CTATAAAATT
181 TTATAATATA GGAAAACTGC TAAATGTAAA TTATACGTTT ACATTTAGCA GTTTATTTT
Length 195 b
Promoter -35- AAGATA (consensus TTGACA)
-10- TATTAT (consensus TATAAT)
Starting point nt 39
Terminator stem loop structure
(rho-independent terminator)
Integration site nt 149
-35 -10 149 39
Features of solB
Secondary structure of solB transcript
Putative mechanism of action of solB transcript
Approaches to improve biological butanol
production
1. Cloning of genes encoding enzymes required for butanol
formation into new host (e. g. E. coli)
This will include all enzymes that convert acetyl-
CoA into butanol (thiolase, 3-hydroxybutyryl-CoA
dehydrogenase, crotonase, butyryl-CoA dehydro-
genase, butyraldehyde dehydrogenase, butanol
dehydrogenase). Important: ETF proteins!
Potential problems:
- expression of enzymes from an anaerobe
- solvent tolerance
2. Tailor-made strain of Clostridium acetobutylicum,
producing only butanol, H2, and CO2
This will be possible by targeted knock-outs of the lactate
dehydrogenase, 2-acetolactate synthase, acetoacetate
decarboxylase, phosphotransacetylase, and phosphotrans-
butyrylase genes, preventing lactate, acetoin, acetone,
acetate, and butyrate formation.
In addition, deregulatory mutations are required to prevent
development of metabolic bottlenecks.
Fermentation substrates
Sugar and starch are excellent substrates for Clostridium
acetobutylicum.
Problems:
1. Limitation of arable land, biofuels from biomass will only
represent a fraction of the total fuel required.
However, this will have a substantial effect on greenhouse
gas emissions.
2. Ethical problem of competition between nutrition and
biofuels.
Possible solutions: Conversion of lignocellulose to sugars or
transfer of butanol production feature to syngas-using bacteria
Commercial Cellulosic Butanol Production
• Cheaper feedstocks & more efficient
fermentation technology required to
improve economics.
• Laihe Rockley Bio-Chemicals resolved to
restart operation of their biobutanol plant
with biomass 150K tonne/year plant in NE China (corn belt)
Largest biobutanol plant in the world
Developed hydrolysis technology for generating
sugars from corn residues
Transitioning (with help grom GBL) from corn
starch to corn residues (bagasse, stover, and
shells).
Green Biologics Ltd., Abingdon, UK:
Industrial technology leader in butanol fermentation
Characteristics:
Gram-positive
obligatly anaerobic
motile
rod (0.6-1 x 2-3 μm)
few endospores
Products:
acetate, ethanol
Doubling times:
fructose (tD = 2,5 h)
synthesis gas (tD = 6-8 h)
1 μm
Clostridium ljungdahlii
Drake et al., 2006. In: The Prokaryotes, 3rd ed., vol. 2, 354-420
CO dehydrogenase/
Acetyl-CoA synthase
Formyl-THF yynthetase
Formate dehydrogenase
Acetyl-CoA
2 e- CO2
CO
Methyl branch Carbonyl branch
Formate Tetrahydrofolate (THF)
Formyl-THF+
ATP
ADP + Pi
Methenyl-THF cylohydrolase
Methenyl-THF
H+
H2O
Methylene-THF dehydrogenase
2 e-
Methylene-THF Methylene-THF reductase
2 e-
Methyl-THF
Methyl-C-FeS-P
C-FeS-P
C-FeS-P
Methyltransferase
[CO] CO
CO dehydrogenase/Acetyl-CoA synthase
HSCoA
Catabolism Anabolism
CO dehydrogenase 2 e-
CO2
2 e-
H2O
(Corrinoid-iron/
sulfur protein)
Wood-Ljungdahl pathway in
C. ljungdahlii
Development of C. ljungdahlii into a novel microbial
production platform from syngas
Genome sequencing and annotation
Suitable shuttle plasmids with antibiotic resistance cassettes (pIMP1,
erythromycin or clarithromycin, thiamphenicol)
Reliable DNA transfer procedure (electroporation, conjugation)
Expression of heterologous genes from other bacteria (clostridia)
butyraldehyde dehydrogenase (AdhE)
butanol dehydrogenase (BdhA)
butyryl-CoA dehydrogenase (Bcd)
crotonase (Crt)
3-hydroxybutyryl-CoA dehydrogenase (Hbd)
thiolase (ThlA)
3-hydroxybutyryl-CoA
crotonyl-CoA
acetyl-CoA acetate acetyl-P acetylaldehyde ethanol
butyryl-CoA
butyraldehyde
butanol
acetoacetyl-CoA
synthesis gas
Butanol synthesis with C. ljungdahlii
pIMP1 pSOBPptb
Butanol production by recombinant C. ljungdahlii
Gene expression in recombinant C. ljungdahlii
Further optimization by "metabolic engineering"
Inactivating genes responsible for butanol degradation
Prohibiting formation of side products by targeted knock-outs
Enhancing butanol formation by increasing plasmid copy number and using stronger promoters
Application vision
Former and current coworkers
in the field presented
Sonja Linder
Niklas Nold
Bettina Schiel-Bengelsdorf
Thiemo Standfest
Kai Thormann
Simone (Lederle) Thum
Brigitte Zickner
Tobias Zimmermann
Financial Support
BMBF-GenoMik/GenoMik-Plus
BMBF-SysMO
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