1. Clostridial fermentations
Clostridia: physiologically and phylogenically diverse group. 14 related families, at least 7 have organisms traditionally called Clostridium (Collins et al. IJSB 44: , 1994)
Genus characteristics:
Gram positive
Spore formers
Butyric acid production.

2. Clostridia Saccharolytic: swollen cells with subterminal spores
C. perfringens group
Intestinal organisms
Toxins, proteases
Sugars or proteins
Butyric acid group
C. butyricum and C. pasteurianium
Solvents by some

3. Clostridia Tetani: amino acid users Putrefactive group C. tetani
Terminal spores
Putrefactive group
Strictly proteolytic
C. sporogenes and C. botulinum
Mercaptans, cadaverine, sulfides

4. Clostridia Substrate-specific groups Acetogens: C. aceticum
Purines: C. acidi-urici, C. cylindrosporum
Ethanol/acetate: C. kluyveri
Acetogens: C. aceticum
Make perdominately acetic acid from sugars
Some use hydrogen and carbon dioxide to make acetic acid
Eubacterium, Peptostreptococcus are intestinal organisms

5. Idealized clostridial fermentation
Glucose
Glycolysis
2 ATP (net)
2 NADH
2 Pyruvates
4 H +
2 CoA
2 Fd ox
Pyruvate-ferredoxin
oxidoreductase
2 Fd
red
2 H 2
2 Acetyl-CoA
+ 2 CO
Hydrogenase 2
Acetyl-CoA
acetyltransferase
CoA O O
Acetoacetyl-CoA CH 3 C CH 2 C
CoA
Β-hydroxybutyryl-CoA
dehydrogenase
NADH OH O
NAD +
3-Hydroxybutyryl-CoA CH 3 CH CH 2 C
CoA
crotonase O H 2 O
Crotonyl-CoA CH 3 CH CH C
CoA
Butyryl-CoA
dehydrogenase
NADH
NAD + O
Butyryl-CoA = CH
phosphotransbutyrylase PO 4 3 CH 2 CH 2 C
CoA
CoA
Butyryl-P
ATP Yield
ADP
Butyrate kinase
used -2
ATP
made 4 + 1
Butyryate
Net= 3 ATP/glucose

6. Claisen condensation to make Acetoacetyl-CoA

7. Pyruvate and Hydrogen Metabolism
Pyruvate:ferredoxin oxidoreductase
Contains
Thiamine pyrophosphate,
But no lipoic acid
Oxidative decarboxylation
Pyruvate
Fdox
CoA
Fdred
CO2
Acetyl-CoA
Ferredoxin: small mol. wt., acidic, protein that acts
as an electron carrier. Has iron-sulfur redox centers
Hydrogen production from pyruvate energetically
favorable at any hydrogen partial pressure.
Pyruvate- + 2H2O -->
acetate - + HCO H+ + 2 H2
∆Go’ = kJ/mol

8. Also, used to reoxidize NADH
Enzyme: hydrogenase
Fdred + 2 H+ --> Fdox + H2
Hydrogen production
Uses reduced ferredoxin as electron donor and
protons as electron acceptor.
Also, used to reoxidize NADH
NADH
NAD+
Fdox
Fdred
2 H+ H2
Hydrogenase
NADH:ferredoxin
oxidoreductase
Net reaction: NADH + 2 H+ --> NAD+ + H2
∆Go’ = + 18 kJ/mol. Only favorable if pH2 is low.

9. Fractionation/Reconstitution
How do we know that ferredoxin is involved in
pyruvate metabolism?
1. Need an assay
Cell-free extract
+ pyruvate = makes H2
- pyruvate = no H2
2. Fractionate and recombine to determine
what components are needed to make H2 from
pyruvate.
Use ion exchange, gel filtration, activated charcoal, etc. to purify.
Mortensen et al., 1962, BBRC 7:
Glass et al J. Bact. 131:

10. Fractionation of extracts by ion exchange
chromatography
Apply cell-free extracts
Wash with buffer
Collect fractions that pass through
column.
Column packed with ion exchange
resin. Proteins with large number
of acidic amino acids will
stick to column.
Collect every 10 ml as
a different fraction and
assay to see if get hydrogen
from pyruvate

11. Assay results
After ion exchange chromatography
Hydrogen from pyruvate?
DEAE-treated
+ pyr no H2
- pyr no H2
DEAE-treated with methyl viologen
+ pyr 6.4 µmol H2
- pyr no H2
Methyl viologen: dye that serves as low potential
electron carrier.
Conclude: something was removed by
chromatography that helps to make hydrogen.

12. Elution of protein on column with
high salt.
Apply high salt to
remove acidic proteins
from column.
Find that a brown-colored
material elutes from
column at 500 mM KCl.
Brown material: shown to be
protein called ferredoxin

13. Assay results: After ion exchange chromatography
and 500 mM KCl treatment.
Hydrogen from pyruvate?
DEAE-treated
+ pyr no H2
- pyr no H2
DEAE-treated with brown material
+ pyr 6.3 µmol H2
- pyr no H2
Addition of ferredoxin restored activity.
Conclude: need ferredoxin to make
hydrogen from pyruvate.

14. Clostridial fermentations
In the idealized case, no acetate was formed since all acetyl-CoA was used to make acetoacetyl-CoA to reoxidize NADH
In real fermentations, clostridia make a mixture of acetate and butyrate.
How can this occur? By having NADH reoxidized by hydrogen production.
Only favorable if hydrogen partial pressure is low

15. Summary Butryic acid formation allows high ATP yield
Claisen condensation: common mechanism in biosynthesis using acyl-CoA substrates.
Pyruvate:Fd oxidoreductase important enzyme in anaerobic metabolism
Hydrogen production from NADH allow fermentative bacteria to maximize ATP gain.
Fractionation/reconstitution is how most pathways were elucidated.