Download - Introduction to Bacterial Fermentations
Egyptian hieroglyphics for henket
http://www.buntesweb.de
History
= beer or cerveza
http://idw-online.de/de/image160582
cuneiform inscription,
Mesopotamia, ≈ 3000 BC
calculations for beer production
Fermentation sensu stricto is an anaerobic process,
i. e. performed by microorganisms in the absence of oxygen
for example beer and wine production by yeast (Saccharomyces cerevisiae)
However, meanwhile also aerobic biotechnological processes are designated as
"fermentation" or "industrial fermention"
for example vinegar production by
Acetobacter aceti (app. 70,000 t/a) or
amino acid production by
Corynebacterium glutamicum
(app. 2.3 million t L-glutamate/a and
1.3 million t L-lysine/a)
Definition of "Fermentation"
L-Glutamate
production
fermenters.
Volume (each):
63,420 gallons;
height: app.
30 m.
Hofu, Japan
(http://smccd.
net/accounts/ca
se/biol230/indfe
r.html)
Industrial products from fermentations
V. Müller, Bacterial fermentation, Encyclopedia of Life Sciences, 2001
Principles of fermentation pathways
V. Müller, Bacterial fermentation, Encyclopedia of Life Sciences, 2001
V. Müller, Bacterial fermentation, Encyclopedia of Life Sciences, 2001
Types of fermentation pathways
Important industrial fermentation
Ethanol fermentation (= bioethanol), Saccharomyces cerevisiae
global production: ≈ 90 million t/a (2012)
beverages: ≈ 18 million t/a
chemical industry: ≈ 7 million t/a
fuel or fuel additive: ≈ 65 million t/a
In Europe, up to 10 % ethanol addition to petrol,
in Brasil up to 85 % or pure ethanol (flexible fuel-engines)
app. 90 % of the global ethanol production stem from the United States and
Brasil.
Substrates for fermentation
Typical substrates for ethanol fermentation are renewables such as
sugar (sugarcane, molasses)(typical for Brazil) or
starch (maize, grain)(typical for the United States, 36 % of
harvest used for fuel ethanol production)
However, maize and grain are also essential for human nutrition.
Competition led to "food vs. fuel" debate.
Massive protests against rising food prices
started in Mexico in 2007 ("tortilla
protests)".
Alternative substrates
Substrates not competing with human nutrition are
lignocellulose hydrolysates (stemming from biomass, collection
and pretreatment required)
gas mixtures (CO2 + H2 or syn(thesis)gas, a mixture of CO + H2)
(industrial waste gases, can also be derived from biomass or
municipal waste)
China prohibited use of starchy substrates, which can serve human
nutrition, for biotechnological production of bulk chemicals and
closed even newly built plants.
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)
Acetogenic organisms
Growth on syngas (less well on CO2 + H2)
very closely related: industrially used by:
Clostridium ljungdahlii acetate, ethanol, butanediol INEOS Bio
Clostridium autoethanogenum acetate, ethanol, butanediol LanzaTech
Clostridium ragsdalei acetate, ethanol, butanediol Coskata
Clostridium coskatii acetate, ethanol, butanediol? Coskata
closely related:
Clostridium carboxidivorans acetate, ethanol, butyrate, butanol Coskata
distantly related:
Clostridium aceticum acetate, no or only little ethanol
Growth on CO2 + H2 (no or poor growth on CO)
Acetobacterium woodii acetate, no or only little ethanol
current industrial use (Clostridium autoethanogenum): ethanol production from steel mill waste
gas
Industrial realization
Ethanol production from steel mill waste gas by C. autoethanogenum (non-recombinant)
Pilot plant, LanzaTech, Demonstration plant, Joint Venture BaoSteel-LanzaTech,
Auckland, NZ Shanghai, China
Acetobacterium woodii
Gram positive
anaerobic
non-sporeforming
39 % G+C content
growth:
chemolithoautotrophically on H2/CO2 or
CO
chemoorganoheterotrophically on e. g.
fructose, glucose, glycerate, lactate
produces acetate
1977 isolated by Balch et al. Balch et al., 1977
CO2
Formate
Formyl-THF
Methyl-THF
Methyl-CoFeS-P Acetyl-CoA Acetyl-P
2 [H]
THF, ATP
Acetate
CO2
2 [H]
2 [H]
2 [H]
H2O
HSCoA
Pi
[CO]
THF CoFeS-P
Acetate production in autotrophic acetogens
Wood-Ljungdahl pathway
ATP
Growth and product pattern of Acetobacterium woodii
on CO2/H2
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A. woodii WT
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A. woodii pJIR750_pta-ack
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Growth and product pattern of Acetobacterium woodii
mutant overexpressing Pta and Ack on CO2/H2
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A. woodii pJIR750_THF
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Growth and product pattern of Acetobacterium woodii
mutant overexpressing THF-dependent enzymes on CO2/H2
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
Sequencing:
- Sanger/pyrosequencing
approach
- 66,585 sequences, up
to 11-fold coverage
- 4,630,065 bp
- 2 putative prophage regions
- no plasmids
Clostridium ljungdahlii: a versatile acetogen
Substrates used
autotrophic growth: H2+CO2, syngas (H2+CO)
heterotrophic growth: fructose, glucose (after adaptation),
gluconate, arabinose, ribose, xylose, erythrose,
threose, formate, pyruvate, malate (pH change
required), fumarate, ethanol, arginine, aspartate,
glutamate, histidine, serine, choline, citrulline,
guanine, hypoxanthine, xanthine
(all as single substrates)
Selected metabolic features
5 Hydrogenases (4 Fe-only, 1 NiFe)
3 Formate dehydrogenases (2 of them selenoenzymes)
2 CO dehydrogenase complexes
Glycolysis, pentose phosphate pathway, no key enzymes of Entner-
Doudoroff pathway
2 Pyruvate:ferredoxin-oxidoreductases
1 Formate:hydrogen-lyase
Branched citrate cycle, leading to fumarate and 2-oxoglutarate
C3 <−−> C4: pyruvate carboxylase, PEP carboxykinase
2 Glycine reductases and a glycine cleavage system
Nitrate (and probably nitrite) reductase
Nitrogenase (Mo-dependent)
Modes of energy conservation during autotrophy
in acetogens
Acetobacterium woodii type:
sodium dependence, formation of
sodium gradient, ATP synthesis via Na+-
ATPase
Moorella thermoacetica type:
cytochromes, menaquinone, formation of
proton gradient, ATP synthesis via H+-
ATPase
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
Characteristics:
Gram-variable
obligatly anaerobic
motile
rod (0.8-1 x 5 μm)
round endospores
Product:
acetate
Doubling times:
fructose (tD = 8 h)
H2/CO2 (tD = 25 h)
Clostridium aceticum
Braun et al., Arch. Microbiol. 128, 288-293 (1981)
Global acetone production: > 5 million tons/year (2011)
Market price: app. 5 billion $/year (2011)
Use:
Acetone as a solvent
Synthesis of cyanohydrin/methylmethacrylate (acrylic glass),
bisphenol, methyisobutylketone, methylisobutylcarbinol,
isophoron
Importance of acetone
acetyl-CoA acetyl-P acetate
CO2
2 [H]
HSCoA
Pi
[CO]
ADP
acetyl-CoA
acetone
CO2
acetoacetate
Adc
acetyl-CoA
acetate
acetoacetyl-CoA
H-S~CoA
ThlA
CO2
formate
formyl-THF
methyl-THF
methyl-CoFeS-P
2 [H]
THF, ATP
2 [H]
2 [H]
H2O
THF CoFeS-P
CtfA/B / AtoDA
H-S~CoA
TEII / YbgC
H2O
Acetone production in acetogenic clostridia
Acetone production from different synthetic operons in C. aceticum
pIMP_adc_ctfAB_thlA pIMP_adc_atoDA_thlA
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pIMP_adc_teII_thlA pIMP_adc_ybgC_thlA
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Acetone production from different synthetic operons in C. aceticum