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Fermentations
384
accumulate superoxide radicals. The superoxide dismutase catalyzes the following reaction:
O2 + O2 + 2H2 H2O2 + O2
Notice that one superoxide radical transfers its
extra electron to the second radical, which is
reduced to hydrogen peroxide. Strict anaerobes
also lack catalase, the enzyme that converts
hydrogen peroxide to water and oxygen:
H2O2 + H2O2 2H2O + O2
Catalase catalyzes the transfer of two electrons
from one hydrogen peroxide molecule to the second, oxidizing the rst to oxygen and reducing
the second to two molecules of water. Table 15.1
shows the distribution of catalase and superoxide dismutase in aerobes and anaerobes.
If the H2O2 is not disposed of, it can oxidize
transition metals such as free iron (II) in the
Fenton reaction and form the free hydroxyl
.
radical, OH :
.
Aerobes or facultative
anaerobes
Escherichia coli
Pseudomonas species
Deinococcus
radiodurans
Aerotolerant bacteria
Butyribacterium
rettgeri
Streptococcus faecalis
Streptococcus lactis
Strict anaerobes
Clostridium
pasteurianum
Clostridium
acetobutylicum
Superoxide
dismutase
Catalase
+
+
+
+
+
+
+
+
fermentations
385
Fig. 15.1 Energy conservation in anaerobic bacteria. (A) Substrate-level phosphorylation: 1, the PGA kinase
reaction; 2, the pyruvate kinase reaction; 3, the acetate kinase reaction. (B) Fumarate respiration. When the
electron donor is NADH, a Q loop probably operates to translocate protons out of the cell. When the electron
donor is periplasmic, proton translocation is not necessary. (C) Efux of an organic acid coupled to protons
or sodium ions (e.g., the coupled efux of protons and lactate by the lactate bacteria). (D) Decarboxylation
of an organic acid coupled to Na+ efux (e.g., Klebsiella). (E) Electrogenic oxalate:formate exchange in
Oxalobacter.
386
acids, alcohols, hydrogen gas, and carbon dioxide. These fermentation end products are oxidized to CO2, H2, and acetate by organisms that
have been only partially identied. Finally, the
methanogens grow on the acetate, H2, and CO2,
converting these to CH4 and CO2.
fermentations
The symbiotic relationship between the obligate proton reducers and the hydrogen utilizers is called a syntrophic association.4 It is also
called interspecies hydrogen transfer. In fact,
many other fermenting bacteria besides the
obligate proton reducers have dehydrogenases
that transfer electrons from NADH to protons
and can use protons as an electron sink when
grown in the presence of hydrogen gas utilizers.
An example is Ruminococcus albus, which is
discussed in Section 15.12.
Hydrogenases are also coupled to the thermodynamically favored oxidation of reduced
ferredoxin, as in pyruvate:ferredoxin oxidoreductase found in the clostridia, sulfate
reducers (Section 13.2.2), and Ruminococcus
albus (see later: Fig. 15.12). Ferredoxins often
have two ironsulfur clusters, each one capable of carrying an electron (also indicated in
Fig. 15.12).
387
388
Substrate
Glucose
Products
Butyrate
Acetate
CO2
H2
Ethanol
Butanol
Acetone
Total
Moles per
100 moles of
substrate
Carbon
(mol)
O/R
value
100
600
4
14
221
135
7
56
22
16
28
221
14
224
66
2
0
+2
1
2
4
2
569
O/R value
(mol/100 mol)
Available
H
Available H
(mol/100 mol)
24
2,400
+442
135
14
224
44
20
8
0
2
12
24
16
80
112
270
84
1,344
352
425, +442
2,242
fermentations
389
Fig. 15.3 Propionate fermentation via the acrylate pathway. Enzymes: 1, lactate dehydrogenase; 2, pyruvate
ferredoxin oxidoreductase; 3, phosphotransacetylase; 4, acetate kinase; 5, CoA transferase; 6, lactyl CoA
dehydratase 7, a dehydrogenase.
390
Fig. 15.4 Propionate fermentation by the succinatepropionate pathway. Enzymes: 1, lactate dehydrogenase (a avoprotein); 2, pyruvate dehydrogenase (an NAD+ enzyme); 3, phosphotransacetylase; 4, acetate
kinase; 5, methylmalonylCoApyruvate transcarboxylase; 6, malate dehydrogenase; 7, fumarase; 8, fumarate reductase; 9, CoA transferase; 10, methylmalonylCoA racemase.
hydrogen for a COSCoA. The enzyme that carries out this reaction is methylmalonylCoA racemase, and it requires vitamin B12 as a cofactor. The
two molecules of methylmalonylCoA donate the
carboxyl groups to pyruvate via the transcarboxylase and in turn become propionylCoA (reaction 5). The propionylCoA donates the CoA to
succinate via the CoA transferase and becomes
propionate (reaction 9). Notice the important role
of transcarboxylases and CoA transferases. These
enzymes allow the attachment of CO2 and CoA to
molecules without the need for ATP.
When comparing Figs. 15.3 and 15.4, we
learn that in metabolism there is sometimes more
than one route to take from point A to point B.
This is demonstrated in the following remarks
about two important enzyme reactions.
1. The fumarate reductase serves as a coupling
site. Propionibacterium and other bacteria
that use the succinatepropionate pathway
use a circuitous route, but one that sends
electrons through an energy-coupling site via
the membrane-bound fumarate reductase.
Electron ow to fumarate requires a quinone, and presumably the p is generated
via a redox loop involving the quinone
(Section 5.6). The use of fumarate as an
electron acceptor during anaerobic growth
is widespread among bacteria. (See the later
discussion of the mixed-acid fermentation in
fermentations
15.7.1 The PEP carboxytransphosphorylase
of propionibacteria and its physiological
significance
The reaction
Propionibacteria growing on carbon sources
such as glucose that enter the glycolytic pathway can produce succinate as well as propionate
as an end product of fermentation. This means
that they must have an enzyme to carboxylate
a C3 intermediate to form the C4 product. The
C3 intermediate that is carboxylated is phosphoenolpyruvate (an intermediate in glycolysis).
The phosphoenolpyruvate is carboxylated to
oxaloacetate, which is then reduced to succinate
via reactions 6, 7, and 8 shown in Fig. 15.4. The
enzyme that catalyzes the carboxylation of phosphoenolpyruvate is called PEP carboxytransphosphorylase, and it catalyzes the following
reaction: (See note 5 for a description of other
C3 carboxylases.)
PEP + CO2 + Pi oxaloacetate + PPi
During the carboxylation, a phosphoryl group
is transferred from PEP to inorganic phosphate
to form pyrophosphate. Pyrophosphate is a
high-energy compound, and propionibacteria
have enzymes that phosphorylate fructose-6phosphate to fructose-1,6-bisphosphate and
serine to phosphoserine, using pyrophosphate
as the phosphoryl donor.
Physiological signicance
The carboxylation of phosphoenolpyruvate or
pyruvate to oxaloacetate and the reduction of
the oxaloacetate to succinate is a widespread
pathway among fermenting bacteria (see, e.g.,
the later discussion of mixed-acid fermentation
in Section 15.10). These reactions were also
391
Fig. 15.5 Acetogenesis from pyruvate by Desulfotomaculum thermobenzoicum. Enzymes: 1, pyruvate dehydrogenase; 2 and 7, phosphotransacetylase; 3 and 8, acetate kinase; 4, enzymes of the acetylCoA pathway; 5
and 6, carbon monoxide dehydrogenase.
392
fermentations
393
Fig. 15.6 Heterofermentative lactate fermentation. Enzymes: 1, hexokinase; 2, glucose-6-phosphate dehydrogenase; 3, 6-phosphogluconate dehydrogenase; 4, ribulose-5-phosphate epimerase; 5, phosphoketolase;
6, phosphotransacetylase; 7, acetaldehyde dehydrogenase; 8, alcohol dehydrogenase; 9, PGALD dehydrogenase; 10, PGA kinase; 11, phosphoglycerate mutase; 12, enolase; 13, pyruvate kinase; 14, lactate dehydrogenase.
Note that the heterofermentative pathway produces only one ATP per glucose in contrast to
the homofermentative pathway, which produces two ATPs for every glucose.
394
fructose-6-phosphate phosphoketolase to
erythrose-4-P and acetylP. The acetylP is
converted to acetate via acetate kinase, with
the formation of an ATP. The erythrose-4-P
reacts with the second fructose-6-P in a transaldolase reaction to form glyceraldehyde-3-P
and sedoheptulose-7-P. These then react in
a transketolase reaction to form xyulose-5-P
and ribose-5-P. The latter isomerizes to form
a second xylulose-5-P. The two xylulose5-P molecules are cleaved by xylulose-5phosphate phosphoketolase to two molecules
of glyceraldehyde-3-P and two of acetylP.
The two glyceraldehyde-3-P molecules are
converted to two lactates with the production
of four ATPs, using reactions of the homofermentative pathway, and the two acetylP
molecules are converted to two acetates with
the production of two more ATPs. Thus, seven
ATPs are produced for every two glucose molecules fermented, but since two ATPs were
used to make the two fructose-6-P molecules,
the net gain in ATP per glucose is 5/2, or 2.5.
Note that since glucose-6-P is not oxidized to
6-phosphogluconate, the acetylP can serve as
a phosphoryl donor for ATP synthesis rather
than being reduced to ethanol.
fermentations
395
Fig. 15.7 Mixed-acid fermentation. Enzymes: 1, glycolytic enzymes; 2, pyruvate kinase; 3, pyruvateformate
lyase; 4, lactate dehydrogenase; 5, formatehydrogen lyase; 6, acetaldehyde dehydrogenase; 7, alcohol dehydrogenase; 8, phosphotransacetylase; 9, acetate kinase; 10, PEP carboxylase; 11, malate dehydrogenase; 12,
fumarase; 13, fumarate reductase. Note the ATP yields: per succinate, approximately 1; per ethanol, 1; per
acetate, 2; per formate, 1; per CO2 and H2, 1; per lactate, 1. Energy equivalent to approximately 1 ATP is
conserved per succinate formed because the fumarate reductase reaction takes place in the cell membrane and
generates a p. Note also the reducing equivalents used in the production of the end products: per succinate,
4; per ethanol, 4; per acetate, 0; per lactate, 2; per formate, 0. The number of reducing equivalents used must
equal the number produced during glycolysis. Therefore, only certain ratios of end products are compatible
with a balanced fermentation.
396
Fig. 15.8 Butanediol formation. Enzymes: 1, glycolytic enzymes; 2, pyruvateformate lyase; 3, formatehydrogen lyase; 4, acetaldehyde dehydrogenase; 5, alcohol dehydrogenase; 6 and 7, -acetolactate synthase; 8,
-acetolactate decarboxylase; 9, 2,3-butanediol dehydrogenase; 10, lactate dehydrogenase.
converted to acetylCoA and formate (reaction 2), and some is used for the synthesis of
2,3-butanediol (reactions 69). The formate
is converted to CO2 and H2 (reaction 3), and
the acetylCoA is reduced to ethanol (reactions 4 and 5). The rst free intermediate in the
butanediol pathway is -acetolactate, formed
by the enzyme -acetolactate synthase, which
decarboxylates pyruvate to enzyme-bound
active acetaldehyde (reaction 6); this is a
reaction that depends upon thiamine pyrophosphate (TPP). The active acetaldehyde is
transferred by the -acetolactate synthase to
pyruvate to form -acetolactate (reaction 7).
The -acetolactate, a -ketocarboxylic acid,
is decarboxylated to acetoin (reaction 8). The
acetoin is reduced by NADH to 2,3-butanediol
(reaction 9). The production of butanediol is
favored under slightly acidic conditions and is
a way for the bacteria to limit the decrease in
external pH caused by the synthesis of organic
acids from pyruvate.
fermentations
397
Fig. 15.9 Proposed mechanism for reactions catalyzed by thiamine pyrophosphate (TPP). Step 1. The TPP enzyme
loses a proton to form a dipolar ion. The anion is stabilized by the positive charge on the nitrogen. The anionic center is nucleophilic and can attack positive centers such as carbonyl carbons. Step 2. The dipolar ion condenses with
pyruvate to form a TPP adduct. Step 3. The electron-attracting N in the TPP facilitates the decarboxylation to form
active acetaldehyde. The active acetaldehyde can form acetaldehyde (step a) or -acetolactate (step b).
398
fermentations
carried out by C. acetobutylicum.9 During exponential growth, in what is called the acidogenic
phase, the bacteria produce butyrate, acetate,
H2, and CO2. When the culture enters stationary
phase, the acids are taken up by the cells, concomitant with the fermentation of the carbohydrate,
399
and are converted to butanol, acetone, and ethanol. This is called the solventogenic phase.
Pentoses are also fermented, and these are
converted to fructose-6-phosphate and phosphoglyceraldehyde via the pentose phosphate
pathway (Fig. 15.11). The pyruvate formed
Fig. 15.11 Butyrate and butanolacetone fermentation in C. acetobutylicum. Carbohydrates are oxidized to
acetylSCoA. Pentose phosphates are converted to fructose-6-phosphate and phosphoglyceraldehyde via the
pentose phosphate reactions. Glucose is oxidized to pyruvate via the EmbdenMeyerhofParnas pathway.
The pyruvate is oxidized to acetylSCoA. In the butyric acid fermentation, the acetylSCoA is converted to
acetate and butyrate (solid lines). When the acetate and butyrate levels rise, they are taken up by the cells and
converted to butanol, and ethanol, while carbohydrates continue to be fermented (dashed lines). During the
butanolacetone fermentation, the acetoacetylSCoA donates the CoASH to butyrate and acetate and becomes
acetoacetate, which is decarboxylated to acetone. Enzymes: 1, pyruvateferredoxin oxidoreductase; 2, acetyl
CoA acetyltransferase (thiolase); 3, hydroxybutyrylCoA dehydrogenase; 4, crotonase; 5, butyrylCoA dehydrogenase; 6, phosphotransbutyrylase; 7, butyrate kinase; 8, phosphotransacetylase; 9, acetate kinase; 10,
acetoacetylSCoA:acetate/butyrate:CoA transferase; 11, acetoacetate decarboxylase; 12, acetaldehyde dehydrogenase; 13, ethanol dehydrogenase; 14, butyraldehyde dehydrogenase; 15, butanol dehydrogenase; 16,
NADHferredoxin oxidoreductase and hydrogenase; 17, hydrogenase. Source: Adapted from Jones, D. T.,
and D. R. Woods. 1986. Acetonebutanol fermentation revisited. Microbiol. Rev. 50:484524.
400
15.13 Summary
Fermentations are cytosolic oxidationreduction pathways in which the electron acceptor is
an organic compound, usually generated in the
pathway.
The source of ATP in fermentative pathways is substrate-level phosphorylation. For
sugar fermentations, these are the phosphoglycerate kinase, pyruvate kinase, acetate
kinase, and butyrate kinase reactions. In other
words, ATP is made from bisphosphoglycerate,
fermentations
401
Fig. 15.12 Fermentation of glucose by Ruminococcus albus. R. albus ferments glucose to a mixture of ethanol, acetate, CO2, and H2. Methanogens draw off the H2, thus stimulating electron ow to H2. The result is a
shift in the fermentation end products toward acetate, accompanied by a greater ATP yield. The production
of H2 by one species and its utilization by another is called interspecies hydrogen transfer. The methanogens
can also utilize the acetate produced by R. albus.
Study Questions
1. Write a fermentation balance using both the
O/R and the available hydrogen method for
the following:
C6H12O6 (glucose) + H2O C2H4O2 (acetate)
+ C2H6O (ethanol)
+ 2H2 + 2CO2
If the EMP pathway is used, what is the
yield of ATP?
402
Lactate
Acetate
Propionate
Succinate
0.31
0.70
0.36
0.61
Lactate
Acetate
Propionate
Formate
Methane
CO2
Product
Selenomonas
Selenomonas +
Methanobacterium
156
46
27
4
0
42
68
99
20
0
51
48
5. Other enzymes besides PEP carboxytransphosphorylase that carboxylate C3 glycolytic intermediates to oxaloacetate are PEP carboxylase and
pyruvate carboxylase (Section 9.9), and PEP carboxykinase (Section 9.13). The latter enzyme usually
operates in the direction of PEP synthesis; however,
in some anaerobes (e.g., Bacteroides) it functions to
synthesize oxaloacetate.
6. Gest, H. 1983. Evolutionary roots of anoxygenic
photosynthetic energy conversion, pp. 215234.
In: The Phototrophic Bacteria: Anaerobic Life in
the Light. Studies in Microbiology, Vol. 4. J. G.
Ormerod (Ed.). Blackwell Scientic Publications,
Oxford.
7. Tasaki, M., Y. Kamagata, K. Nakamura, K.
Okamura, and K. Minami. 1993. Acetogenesis from
pyruvate by Desulfotomaculum thermobenzoicum
and differences in pyruvate metabolism among three
sulfate-reducing bacteria in the absence of sulfate.
FEMS Microbiol. Lett. 106:259264.
8. Gas production is generally observed as a bubble
in an inverted vial placed in the fermentation tube.
The bubble is due to H2, since CO2 is very soluble in
water.
9. Jones, D. T., and D. R. Woods. 1986. Acetone
butanol fermentation revisited. Microbiol. Rev.
50:484524.
10. Hamilton, W. A. 1988. Energy transduction in
anaerobic bacteria, pp. 83149. In: Bacterial Energy
Transduction. C. Anthony (Ed.). Academic Press,
New York.