introduction to bacterial fermentations

Introduction to Bacterial Fermentations

Peter Dürre, University of Ulm Valencia, November 8, 2013

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



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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acetate

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A. woodii pJIR750_pta-ack

growth

acetate

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mutant overexpressing Pta and Ack on CO2/H2

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A. woodii pJIR750_THF

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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

Hypothesis: generation of proton gradient via Rnf system

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

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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Acetone production from different synthetic operons in C. aceticum

Application vision

Former and current coworkers

in the field presented

Frank Bengelsdorf

Catarina Erz

Sebastian Flüchter

Sabrina Hoffmeister

Michael Köpke

Bettina Schiel-Bengelsdorf

Melanie Straub

Simone (Lederle) Thum

Brigitte Zickner

Tobias Zimmermann

Financial Support

BMBF-GenoMik/GenoMik-Plus

BMBF-SysMO

introduction to bacterial fermentations

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