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Fermentations
383
384 the physiology and biochemistry of prokaryotes
accumulate superoxide radicals. The superox- bacteria reveals a variety of methods, which
ide dismutase catalyzes the following reaction: were described in earlier chapters. Some of these
are reviewed in Fig. 15.1. Most fermenting bac-
O2 + O2 + 2H2 H2O2 + O2
teria make most or all of their ATP via substrate-
Notice that one superoxide radical transfers its level phosphorylations, and they create a p
extra electron to the second radical, which is (needed for solute transport, motility, etc.) by
reduced to hydrogen peroxide. Strict anaerobes reversing the membrane-bound ATPase. Many
also lack catalase, the enzyme that converts anaerobic bacteria also generate a p by reduc-
hydrogen peroxide to water and oxygen: ing fumarate. This is an anaerobic respiration
that can occur during fermentative metabolism
H2O2 + H2O2 2H2O + O2
when fumarate is produced. During fumar-
Catalase catalyzes the transfer of two electrons ate reduction, NADH dehydrogenases donate
from one hydrogen peroxide molecule to the sec- electrons to fumarate via menaquinone, and a
ond, oxidizing the rst to oxygen and reducing p is created, perhaps via a quinone loop. (See
the second to two molecules of water. Table 15.1 Section 5.6.1 for a discussion of quinone loops.)
shows the distribution of catalase and superox- The production of a p by fumarate respiration
ide dismutase in aerobes and anaerobes. in fermenting bacteria probably spares ATP
If the H2O2 is not disposed of, it can oxidize that would normally be hydrolyzed to maintain
transition metals such as free iron (II) in the the p.
Fenton reaction and form the free hydroxyl Some anaerobes (e.g., Wolinella succino-
.
radical, OH : genes) carry out a periplasmic oxidation of
. electron donors, such as H2 or formate in the
Fe2+ + H+ + H2O2 Fe3+ + OH + H2O
case of W. succinogenes, to create a p. (See
(See note 3 for a denition of transition metals.) Section 5.7.4.) There are several other means
available to anaerobic bacteria for generating a
15.2 Energy Conservation by proton motive force or a sodium motive force.
Anaerobic Bacteria Some anaerobes and facultative anaerobes are
capable of creating an electrochemical ion gra-
An examination of mechanisms of energy con-
dient by symport of organic acids out of the
servation and ATP production in anaerobic
cell with protons or sodium ions. The organic
acids are produced during fermentation, and
Table 15.1 The distribution of catalase and
the energy to create the electrochemical gradi-
superoxide dismutase ent is due to the concentration gradient of the
excreted organic acid (high inside). This has
Bacterium Superoxide Catalase been demonstrated for lactate excretion (pro-
dismutase ton symport) by the lactate bacteria and for
succinate excretion (sodium ion symport) by a
Aerobes or facultative
anaerobes rumen bacterium, Selenomonas ruminantium.
Escherichia coli + + (See Section 4.8.3 for a discussion of lactate and
Pseudomonas species + + succinate efux in symport with protons and
Deinococcus + + sodium ions.)
radiodurans
Klebsiella pneumoniae is capable of generat-
Aerotolerant bacteria
Butyribacterium + ing an electrochemical sodium ion gradient by
rettgeri using the energy released from the decarboxyla-
Streptococcus faecalis + tion of oxaloacetate to pyruvate (Section 4.8.1).
Streptococcus lactis + The decarboxylase pumps Na+ out of the cell.
Strict anaerobes
The sodium potential that is created is used to
Clostridium
pasteurianum drive the uptake of oxaloacetate, which is used
Clostridium as a carbon and energy source. Oxalobacter
acetobutylicum formigenes creates a proton potential by cata-
Source: Stanier, R. Y., J. L. Ingraham, M. L. Wheelis, and lyzing an electrogenic anion exchange coupled
P. R. Painter. 1986. The Microbial World. Reprinted by per- to the decarboxylation of oxalate to formate
mission of Prentice-Hall, Upper Saddle River, NJ. (Section 4.8.2).
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.
The symbiotic relationship between the obli- that the C/O ratio is 1. This changes C2H6O
gate proton reducers and the hydrogen utiliz- to C2H8O2. Acetic acid is C2H4O2. Since the
ers is called a syntrophic association.4 It is also C/O ratio is already 1, nothing further need
called interspecies hydrogen transfer. In fact, be done. The formula for carbon dioxide is
many other fermenting bacteria besides the CO2. To make the C/O ratio 1, it is necessary
obligate proton reducers have dehydrogenases to subtract H2O, that is: [CO2 H2O]. The
that transfer electrons from NADH to protons result is C(2H)O.
and can use protons as an electron sink when 2. Now compare the number of hydrogens in
grown in the presence of hydrogen gas utilizers. the modied formula with (CH2O)n, which
An example is Ruminococcus albus, which is has the same number of carbons as in the
discussed in Section 15.12. modied formula. For ethanol, C2H8O2 is
Hydrogenases are also coupled to the ther- compared with (CH2O)2.There are 8 hydro-
modynamically favored oxidation of reduced gens in C2H8O2 but only 4 in (CH2O)2. Thus,
ferredoxin, as in pyruvate:ferredoxin oxi- ethanol has an additional 4 hydrogens.
doreductase found in the clostridia, sulfate Carbon dioxide, C(2H)O, however, has
reducers (Section 13.2.2), and Ruminococcus 4 fewer hydrogens than CH2O. Acetic acid
albus (see later: Fig. 15.12). Ferredoxins often (C2H4O2) and (CH2O)2 have the same num-
have two ironsulfur clusters, each one capa- ber of hydrogens.
ble of carrying an electron (also indicated in 3. Add 1 for each additional 2H and +1 for a
Fig. 15.12). decrease in 2H. Thus, the O/R for ethanol is
2, for CO2 it is +2, and for acetic acid it is 0.
15.5 How to Balance a Fermentation Since both the oxidized and reduced fermenta-
A written fermentation is said to be balanced tion end products originate from the substrate,
when the hydrogens produced during the oxi- in a balanced fermentation, the sum of the O/R
dations equal the hydrogens transferred to the of the products equals the O/R of the substrate.
fermentation end products. Only under these For example, the O/R for glucose is 0. When
conditions can all of the NADH and reduced one mole of glucose is fermented to two moles
ferredoxin be recycled to the oxidized forms. It of ethanol and two moles of carbon dioxide, the
is important to know whether a fermentation O/R of the products is (2 2) + (+2 2) = 0.
is balanced because if it is not, the overall writ- Often one simply takes the ratio (+/) of the
ten reaction is incorrect. There are two methods O/R of the products when a carbohydrate is
used to balance fermentations, the oxidation/ fermented. For a balanced fermentation, +/
reduction (O/R) method and the available should be 1.
hydrogen method; both are bookkeeping meth-
ods to keep track of the hydrogens.
15.5.2 The available hydrogen method
Like the O/R method, the available hydrogen
15.5.1 The O/R method
procedure is merely one of bookkeeping. It has
The O/R method entailing the computation of nothing to do with the chemistry of the reac-
an oxidation/reduction balance is computed as tions. According to this method, one oxidizes
described here. One arbitrarily designates as the molecule to CO2 by using water to obtain
zero the O/R value for formaldehyde (CH2O) the available hydrogen. For example:
and multiples thereof [(CH2O)n] and uses that
formula as a standard against which to com- C6H12O6 + 6H2O 24H + 6CO2
pare the reduction level of other molecules.
Thus glucose has 24 available hydrogens.
Following are the steps in determining the O/R
The available H in all the products must add
value of any molecule.
up to the available H in the starting material.
1. Add or subtract water to the molecule in Table 15.2 gives the concentration of products
question to make the C/O ratio 1. This will per 100 moles of glucose used. The available H
allow a comparison to (CH2O)n. For exam- in the glucose is 24 100 = 2,400. The available
ple, the formula for ethanol is C2H6O. The H in the products adds up to 2,242. Thus, the
C/O ratio is 2. One water must be added so balance is 2,400/2,242 = 1.07.
388 the physiology and biochemistry of prokaryotes
Substrate
Glucose 100 600 0 24 2,400
Products
Butyrate 4 16 2 8 20 80
Acetate 14 28 0 8 112
CO2 221 221 +2 +442 0
H2 135 1 135 2 270
Ethanol 7 14 2 14 12 84
Butanol 56 224 4 224 24 1,344
Acetone 22 66 2 44 16 352
Total 569 425, +442 2,242
Source: Gottschalk, G. 1986. Bacterial Metabolism. Springer-Verlag, Berlin.
15.6 Propionate Fermentation via yielding 2[H] again (reaction 2). The acetyl
the Acrylate Pathway CoA is converted to acetate and ATP via
acetylP (reactions 3 and 4). During the oxida-
The genus Clostridium comprises a heteroge-
tions, 4[H] are produced, which must be reuti-
neous group of bacteria consisting of gram-
lized. The electron acceptor is created from a
positive, anaerobic, spore-forming bacteria
second and third molecule of lactate (actually
that cannot use sulfate as a terminal electron
lactylCoA). The lactate acquires a CoA from
acceptor. The clostridia can be isolated from
propionylCoA (reaction 5). The lactylCoA is
anaerobic regions (or areas of low oxygen lev-
dehydrated to yield the unsaturated molecule,
els) in soil. They ferment organic nutrients to
acrylylCoA (reaction 6). Each acrylylCoA
products that can include alcohols, organic
is then reduced to propionylCoA, using up
acids, hydrogen gas, and carbon dioxide. C.
the 4[H] (reaction 7). The fermentation is thus
propionicum oxidizes three moles of lactate to
balanced. The propionate, which is produced
two moles of propionate, one mole of acetate,
during the CoA transfer step (reaction 5), is
and one mole of carbon dioxide, to produce one
catalyzed by CoA transferase, an enzyme that
mole of ATP. The pathway is called the acry-
occurs in many anaerobes.
late pathway because one of the intermediates
What can we learn from this pathway? The
is acrylylCoA. The bacteria derive ATP via
bacteria use a standard method for making ATP
a substrate-level phosphorylation during the
under anaerobic conditions. They decarboxy-
conversion of acetylP to acetate catalyzed by
late pyruvate to acetylCoA and then, using a
acetate kinase. Since only one acetate is made
phosphotransacetylase (to make acetylP) and
for every three lactates used, the pathway yields
an acetate kinase, they make ATP and acetate.
one-third of an ATP per lactate. Growth yields
These reactions are widespread among fer-
are proportional to the amount of ATP pro-
menting bacteria. The production of acetate is
duced, and it is to be expected that the growth
presumed to be associated always with the syn-
yields for these organisms are very low. (The
thesis of two moles of ATP per mole of acetate
molar growth yield for ATP is 10.5 g of cells per
if the bacteria are growing on glucose and using
mole of ATP synthesized, Section 2.3.)
the EmbdenMeyerhofParnas pathway. One
ATP is produced from acetylCoA, and the sec-
15.6.1 The fermentation pathway of ond ATP is produced during the production of
Clostridium propionicum the pyruvate in the EMP pathway.
A molecule of lactate is oxidized to pyruvate, Another common reaction among ferment-
yielding 2[H] (Fig. 15.3, reaction 1). The pyru- ing bacteria is the transfer of coenzyme A from
vate is then oxidized to acetylCoA and CO2, one organic molecule to another, a reaction
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.
catalyzed by CoA transferase. The other way of characteristic avor of this cheese is due to the
attaching a coenzyme A molecule to a carboxyl propionate, and the holes in the cheese are due
group is to transfer an AMP or a phosphate to to the carbon dioxide produced.
the carboxyl group from ATP, making an acyl The pathway illustrated in Fig. 15.4 shows
phosphate or an acylAMP, and then displac- that three molecules of lactate are oxidized to
ing the AMP or phosphate with CoASH. (Recall pyruvate (reaction 1). This yields six electrons.
the activation of fatty acids prior to their deg- Then, one pyruvate is oxidized to acetate, CO2,
radation, Section 10.1.1.) However, fermenting and ATP, yielding two more electrons (reac-
organisms must conserve ATP. The CoA trans- tion 2). We now have eight electrons to use up.
ferase reaction is one way this can be done. The other two pyruvates are carboxylated to
yield two molecules of oxaloacetate (reaction 5).
The reaction is catalyzed by methylmalonyl
15.7 Propionate Fermentation via CoA transcarboxylase. The two oxaloacetates
the SuccinatePropionate Pathway are reduced to two malates, consuming a total
Many bacteria that yield propionic acid as a of four electrons (reaction 6). The two malates
product of fermentation use a pathway dif- are dehydrated to two fumarates (reaction 7).
ferent from the acrylate pathway. The other The two fumarates are reduced via fumarate
pathway, called the succinatepropionate reductase to two succinates, consuming four
pathway, yields more ATP than the acrylate electrons (reaction 8). The latter reduction is
pathway per mole of propionate formed. One coupled to the generation of a p. The fermen-
of the organisms that utilizes this pathway, tation is now balanced.
Propionibacterium, ferments lactate as well as The two succinates are converted to two mol-
hexoses to a mixture of propionate, acetate, ecules of succinylCoA via a CoA transferase
and CO2. Propionibacterium is a gram-positive (reaction 9). The two molecules of succinyl
anaerobic, nonsporulating, nonmotile, pleo- CoA are isomerized to two molecules of meth-
morphic rod that is part of the normal ora ylmalonylCoA in an unusual reaction in which
in the rumen of herbivores; it is also found on COSCoA moves from the carbon to the car-
human skin and in dairy products (e.g., cheese). bon in succinylCoA to form methylmalonyl
Propionibacterium is used in the fermenta- CoA (reaction 10). The reaction can be viewed
tion process that produces Swiss cheese. The as an exchange between adjacent carbons of a
390 the physiology and biochemistry of prokaryotes
Fig. 15.4 Propionate fermentation by the succinatepropionate pathway. Enzymes: 1, lactate dehydroge-
nase (a avoprotein); 2, pyruvate dehydrogenase (an NAD+ enzyme); 3, phosphotransacetylase; 4, acetate
kinase; 5, methylmalonylCoApyruvate transcarboxylase; 6, malate dehydrogenase; 7, fumarase; 8, fumar-
ate reductase; 9, CoA transferase; 10, methylmalonylCoA racemase.
hydrogen for a COSCoA. The enzyme that car- Section 15.10.) The electron donors besides
ries out this reaction is methylmalonylCoA race- lactate include NADH, H2, formate, and
mase, and it requires vitamin B12 as a cofactor. The glycerol-3-phosphate. The p that is estab-
two molecules of methylmalonylCoA donate the lished can be used for ATP synthesis, for sol-
carboxyl groups to pyruvate via the transcarbox- ute uptake, or to spare ATP that might be
ylase and in turn become propionylCoA (reac- hydrolyzed to maintain the p.
tion 5). The propionylCoA donates the CoA to 2. The transcarboxylase reaction spares an
succinate via the CoA transferase and becomes ATP. One way to carboxylate pyruvate
propionate (reaction 9). Notice the important role is to use pyruvate carboxylase and CO2
of transcarboxylases and CoA transferases. These (Section 9.9). However, this requires an
enzymes allow the attachment of CO2 and CoA to ATP. Propionibacterium has a transcarbox-
molecules without the need for ATP. ylase called methylmalonylCoApyruvate
When comparing Figs. 15.3 and 15.4, we transcarboxylase that transfers a carboxyl
learn that in metabolism there is sometimes more group from methylmalonylCoA to pyru-
than one route to take from point A to point B. vate, hence an ATP is not used (Fig. 15.4,
This is demonstrated in the following remarks reaction 5). By using the transcarboxylase,
about two important enzyme reactions. the bacteria save energy by substituting one
covalent bond for another. However, not all
1. The fumarate reductase serves as a coupling fermenting bacteria that produce propionate
site. Propionibacterium and other bacteria from pyruvate use the methylmalonylCoA
that use the succinatepropionate pathway pyruvate transcarboxylase. For example,
use a circuitous route, but one that sends Veillonella alcalescens and Propionigenum
electrons through an energy-coupling site via modestum use a sodium-dependent decar-
the membrane-bound fumarate reductase. boxylase to remove the carboxyl group
Electron ow to fumarate requires a qui- from methylmalonylCoA while generat-
none, and presumably the p is generated ing an electrochemical potential (Section
via a redox loop involving the quinone 4.8.1). The sodium-dependent decarboxy-
(Section 5.6). The use of fumarate as an lase pumps sodium ions out of the cell, gen-
electron acceptor during anaerobic growth erating a sodium ion potential, which can be
is widespread among bacteria. (See the later used as a source of electrochemical energy
discussion of the mixed-acid fermentation in (e.g., for solute uptake or ATP synthesis).
fermentations 391
15.7.1 The PEP carboxytransphosphorylase discussed in the context of the reductive citric
of propionibacteria and its physiological acid pathway (Section 9.8).
significance The pathway from PEP or pyruvate to suc-
The reaction cinate is extremely important for anaerobes
Propionibacteria growing on carbon sources and serves three purposes: (1) the fumarate is an
such as glucose that enter the glycolytic path- electron sink, enabling NADH to be reoxidized;
way can produce succinate as well as propionate (2) the fumarate reductase is a coupling site (i.e.,
as an end product of fermentation. This means a p is generated); and (3) the succinate can be
that they must have an enzyme to carboxylate converted to succinylCoA, which is required
a C3 intermediate to form the C4 product. The for the biosynthesis of tetrapyrroles, lysine,
C3 intermediate that is carboxylated is phospho- diaminopimellic acid, and methionine. With
enolpyruvate (an intermediate in glycolysis). respect to fumarate acting as an electron sink,
The phosphoenolpyruvate is carboxylated to Gest has suggested that the carboxylation of
oxaloacetate, which is then reduced to succinate the C3 glycolytic intermediate and the reductive
via reactions 6, 7, and 8 shown in Fig. 15.4. The pathway from oxaloacetate to succinate may
enzyme that catalyzes the carboxylation of phos- have evolved when the earths atmosphere was
phoenolpyruvate is called PEP carboxytrans- still anaerobic, serving the purpose of balancing
phosphorylase, and it catalyzes the following fermentations and thus sparing one of the two
reaction: (See note 5 for a description of other pyruvates derived from glucose for biosynthe-
C3 carboxylases.) sis.6 The reactions between oxaloacetate and
succinate may have become part of the oxida-
PEP + CO2 + Pi oxaloacetate + PPi tive citric acid cycle later, during the evolution
During the carboxylation, a phosphoryl group of aerobic metabolism.
is transferred from PEP to inorganic phosphate
to form pyrophosphate. Pyrophosphate is a
high-energy compound, and propionibacteria
15.8 Acetate Fermentation
have enzymes that phosphorylate fructose-6- (Acetogenesis)
phosphate to fructose-1,6-bisphosphate and As discussed in Section 14.1.3 in connection
serine to phosphoserine, using pyrophosphate with the acetogenic bacterium Clostridium
as the phosphoryl donor. thermoaceticum, some bacteria use CO2 as an
electron sink and reduce it to acetate as a fer-
Physiological signicance mentation end product via the acetylCoA
The carboxylation of phosphoenolpyruvate or pathway. This is called acetogenesis. Another
pyruvate to oxaloacetate and the reduction of acetogenic bacterium is the sulfate reducer
the oxaloacetate to succinate is a widespread Desulfotomaculum thermobenzoicum, when it
pathway among fermenting bacteria (see, e.g., is growing on pyruvate in the absence of sulfate.7
the later discussion of mixed-acid fermentation (Another sulfate reducer, Desulfobulbus propi-
in Section 15.10). These reactions were also onicus, uses the succinatepropionate pathway
Fig. 15.5 Acetogenesis from pyruvate by Desulfotomaculum thermobenzoicum. Enzymes: 1, pyruvate dehy-
drogenase; 2 and 7, phosphotransacetylase; 3 and 8, acetate kinase; 4, enzymes of the acetylCoA pathway; 5
and 6, carbon monoxide dehydrogenase.
392 the physiology and biochemistry of prokaryotes
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 dehy-
drogenase; 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.
9). The formate can be oxidized to CO2 and H2 fermentation, two electrons must be used for
by the enzyme system formatehydrogen lyase each phosphoenolpyruvate or pyruvate formed.
(reaction 5). This system actually consists of The pathways that utilize four electrons per
two enzymes; formate dehydrogenase and an phosphoenolpyruvate or pyruvate formed spare
associated hydrogenase. The formate dehydro- the second phosphoenolpyruvate or pyruvate
genase oxidizes the formate to CO2 and reduces for biosynthesis. However, they may do this at
the hydrogenase, which transfers the electrons the expense of an ATP. For example, the reduc-
to two protons to form hydrogen gas. Shigella tion of acetylCoA to ethanol uses 4H, but this
and Erwinia do not contain formatehydrogen is at the expense of an ATP that can be formed
lyase, and therefore do not produce gas. (See when acetylCoA is converted to acetate. In this
note 8.) context, the production of succinate is particu-
Each of the pathways following phosphoe- larly valuable. The pathway utilizes 4H and also
nolpyruvate or pyruvate can be viewed as a met- includes a coupling site (fumarate reductase).
abolic branch that accepts different amounts of
reducing equivalent, that is, 0, 2[H], or 4[H], 15.10.2 Butanediol fermentation
depending upon the pathway. The reducing The butanediol fermentation is character-
equivalents in the different branches are succi- ized by the production of 2,3-butanediol and
nate (4), ethanol (4), lactate (2), acetate (0), and acetoin (Fig. 15.8). Glucose is oxidized via the
formate (0). During glycolysis, two electrons glycolytic pathway to pyruvate (reactions 1).
are produced for each phosphoenolpyruvate There are three fates for the pyruvate. Some of
or pyruvate formed. Therefore, to balance the it is reduced to lactate (reaction 10), some is
396 the physiology and biochemistry of prokaryotes
Fig. 15.8 Butanediol formation. Enzymes: 1, glycolytic enzymes; 2, pyruvateformate lyase; 3, formatehy-
drogen 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 (reac- How thiamine pyrophosphate catalyzes the
tion 2), and some is used for the synthesis of decarboxylation of -ketocarboxylic acids
2,3-butanediol (reactions 69). The formate The decarboxylation of -ketocarboxylic
is converted to CO2 and H2 (reaction 3), and acids presents a problem because there is no
the acetylCoA is reduced to ethanol (reac- electron-attracting carbonyl group to the
tions 4 and 5). The rst free intermediate in the CC bond to withdraw electrons, as there is
butanediol pathway is -acetolactate, formed in -ketocarboxylic acids. (See Section 9.11.2
by the enzyme -acetolactate synthase, which for a description of the decarboxylation of
decarboxylates pyruvate to enzyme-bound -ketocarboxylic acids.) The problem is solved
active acetaldehyde (reaction 6); this is a by using thiamine pyrophosphate (TPP). A pro-
reaction that depends upon thiamine pyro- posed mechanism is illustrated in Fig. 15.9. A
phosphate (TPP). The active acetaldehyde is proton dissociates from the thiamine pyrophos-
transferred by the -acetolactate synthase to phate to form a dipolar ion, which is stabilized
pyruvate to form -acetolactate (reaction 7). by the positive charge on the nitrogen atom.
The -acetolactate, a -ketocarboxylic acid, The negative center of the dipolar ion adds to
is decarboxylated to acetoin (reaction 8). The the keto group of the -ketocarboxylic acid.
acetoin is reduced by NADH to 2,3-butanediol Then, the electron-attracting =N+ group pulls
(reaction 9). The production of butanediol is electrons away from the CC bond, facilitat-
favored under slightly acidic conditions and is ing the decarboxylation. The product is an
a way for the bacteria to limit the decrease in -ketol called active aldehyde. The ow
external pH caused by the synthesis of organic of electrons is reversed, and the active alde-
acids from pyruvate. hyde then attacks a positive center on another
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 cen-
ter 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).
carried out by C. acetobutylicum.9 During expo- and are converted to butanol, acetone, and etha-
nential growth, in what is called the acidogenic nol. This is called the solventogenic phase.
phase, the bacteria produce butyrate, acetate, Pentoses are also fermented, and these are
H2, and CO2. When the culture enters stationary converted to fructose-6-phosphate and phos-
phase, the acids are taken up by the cells, concom- phoglyceraldehyde via the pentose phosphate
itant with the fermentation of the carbohydrate, 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 dehy-
drogenase; 6, phosphotransbutyrylase; 7, butyrate kinase; 8, phosphotransacetylase; 9, acetate kinase; 10,
acetoacetylSCoA:acetate/butyrate:CoA transferase; 11, acetoacetate decarboxylase; 12, acetaldehyde dehy-
drogenase; 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 the physiology and biochemistry of prokaryotes
from the sugars is oxidatively decarboxylated to mixed populations can inuence fermentation
acetylCoA and CO2 via the pyruvateferredoxin end products. The fermentation is easy to under-
oxidoreductase (reaction 1). The acetylCoA stand. The glucose is rst oxidized to two moles
has two fates. Some of it is converted to butyrate of pyruvate, yielding two moles of NADH. Then
and ATP as described in Fig. 15.10 (reactions each mole of pyruvate is oxidized to acetylCoA,
27). Some acetylCoA is also converted to CO2, and H2 by means of the pyruvateferre-
acetate via acetylP in a reaction that yields an doxin oxidoreductase and hydrogenase. One of
additional ATP (Fig. 15.11, reactions 8 and 9). the acetylCoA molecules is converted to acetate,
The ability to send electrons to hydrogen via the allowing an ATP to be made. The second acetyl
NADH:ferredoxin oxidoreductase (reaction 16) CoA is reduced to ethanol via the two NADHs.
allows the bacteria to produce more acetate, Thus the fermentation is balanced. The overall
hence more ATP, rather than reducing acetyl reaction is the conversion of one mole of glucose
CoA to butyrate. Notice that the reaction gener- to one mole of ethanol, one mole of acetate, two
ates twice as much ATP as butyrate per acetate. moles of hydrogen, and two moles of carbon
However, reduction of protons by NADH is not dioxide yielding, three ATPs.
favored energetically and is limited by increasing Something different happens when the bacte-
concentrations of hydrogen gas. rium is grown in a coculture with a methanogen.
The NADH oxidoreductase can be pulled in the Growth with a methanogen shifts the fermenta-
direction of hydrogen gas by other bacteria that tion in the direction of acetate with the concomi-
utilize hydrogen in a process called interspecies tant production of more ATP. This is explained
hydrogen transfer, explained in Section 15.4.1. In in the following way. R. albus has a hydrogenase
the solventogenic phase, butyrate and acetate are that transfers electrons from NADH to H+ to
taken up by the cells and reduced to butanol and produce hydrogen gas. When hydrogen accu-
ethanol (dashed lines in Fig. 15.11). The acids mulates in the medium, the hydrogenase does
are converted to their CoA derivatives by accept- not oxidize NADH because the equilibrium
ing a CoA from acetoacetylCoA (reaction 10). favors NAD+ reduction. The NADH therefore
The reaction is catalyzed by CoA transferase. reduces acetylCoA to ethanol. However, the
(Recall that CoA transferase is also used in the methanogen utilizes the hydrogen gas for meth-
acrylate pathway for propionate fermentation, ane production and keeps the hydrogen levels
Section 15.6.) The acetoacetate that is formed is very low. In the presence of the methanogen, the
decarboxylated to acetone and CO2 (reaction 11). NADH in R. albus reduces protons to hydrogen
The acetylCoA is reduced to ethanol (reactions gas instead of reducing acetylCoA. The result is
12 and 13), and the butyrylCoA is reduced to that R. albus converts the acetylCoA to acetate.
butanol (reactions 14 and 15). This is also an advantage to the methanogen,
The molar ratios of the fermentation end prod- since methanogens can also use acetate as a car-
ucts in clostridial fermentations will vary accord- bon and energy source. The transfer of hydrogen
ing to the strain.10 For example, C. acetobutylicum gas from one species to another is called inter-
is an important solvent-producing strain, and species hydrogen transfer and is an example of
when grown at pH0 below 5 it produces butanol nutritional synergism found among mixed pop-
and acetone in the molar ratio of 2:1 with small ulations of bacteria. (See the discussion of inter-
amounts of isopropanol, whereas C. sporogenes species hydrogen transfer in Section 15.4.1.)
and C. pasteurianum produce very little solvent.
Clostridium butyricum forms butyrate and ace- 15.13 Summary
tate in a ratio of about 2:1, whereas C. perfrin-
Fermentations are cytosolic oxidationreduc-
gens produces these acids in a ratio of 1:2, with
tion pathways in which the electron acceptor is
signicant amounts of ethanol and lactate.
an organic compound, usually generated in the
pathway.
15.12 Ruminococcus albus The source of ATP in fermentative path-
Ruminococcus albus is a rumen bacterium ways is substrate-level phosphorylation. For
that can use the glycolytic pathway to ferment sugar fermentations, these are the phospho-
glucose to ethanol, acetate, H2, and CO2 (Fig. glycerate kinase, pyruvate kinase, acetate
15.12). It is a good fermentation to examine kinase, and butyrate kinase reactions. In other
because it illustrates how growth of bacteria in 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 etha-
nol, 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.
phosphoenolpyruvate, acetylP, and butyrylP. pyruvate plus CO2. Protons can also be used
ButyrylP is a phosphoryl donor during butyrate as electron sinks, and many fermenting bacte-
fermentations. ria have hydrogenases that reduce protons to
However, other means of conserving energy hydrogen gas.
are available for fermenting bacteria. For exam- The excreted end products of fermentations,
ple, a p can be created during electron ow to including hydrogen gas, are used by other
fumarate, the fumarate being generated during anaerobic bacteria so that an anaerobic food
fermentation. Other means of creating a proton chain develops. At the bottom of the food chain
potential or sodium potential exist in certain are the methanogens, which convert hydrogen
groups of bacteria. These include efux of car- gas, carbon dioxide, and acetate to CO2 and
boxylic acids in symport with protons or sodium CH4. These are recycled to organic carbon in
ions, decarboxylases that function as sodium the biosphere for use by autotrophic and metha-
ion pumps, and oxalate:formate exchange. notrophic organisms as a source of carbon.
Besides making ATP, fermenting bacte-
ria must have a place to unload the electrons Study Questions
removed during oxidation reactions. Of course,
the reason for this is that they must regenerate 1. Write a fermentation balance using both the
the NAD+, oxidized ferredoxin, and FAD to O/R and the available hydrogen method for
continue the fermentation. The electron accep- the following:
tors are sometimes referred to as electron
C6H12O6 (glucose) + H2O C2H4O2 (acetate)
sinks. During a fermentation the electron sinks
+ C2H6O (ethanol)
are created from the carbon source. In fact, all
+ 2H2 + 2CO2
the electron sinks for the major carbohydrate
fermentations either are pyruvate itself or are If the EMP pathway is used, what is the
synthesized from pyruvate or phosphoenol yield of ATP?
402 the physiology and biochemistry of prokaryotes
3. Use the EntnerDoudoroff pathway to 1. Imlay, J. A. 2008. Cellular defenses against super-
write an ethanol fermentation. Contrast oxide and hydrogen peroxide. Annu. Rev. Biochem.
77:755776.
the ATP yields with an ethanol fermenta-
tion by means of the EMP pathway. 2. Henle, E. S., and S. Linn. 1997. Formation, pre-
vention, and repair of DNA damage by iron/hydro-
4. Consider the following fermentation data gen peroxide. J. Biol. Chem. 272:1909519098.
for Selenomonas ruminantium products 3. Transition elements are dened as those elements
formed per millimole of glucose: in which electrons are added to an outer electronic
shell before one of the inner shells is complete. There
Product Amount formed (nmol) are nine of them. They are all metals, and they include
iron, which is the one that occurs in highest amounts
Lactate 0.31 in living systems.
Acetate 0.70 4. Shink, B. 1997. Energetics of syntrophic coopera-
Propionate 0.36 tion in methanogenic degradation. Microbiol. Mol.
Succinate 0.61 Biol. Rev. 61:262280.
Source: Michel, T. A., and J. M. Macy. 1990. 5. Other enzymes besides PEP carboxytransphos-
Generation of a membrane potential by sodium- phorylase that carboxylate C3 glycolytic interme-
dependent succinate efux in Selenomonas diates to oxaloacetate are PEP carboxylase and
ruminantium. J. Bacteriol. 172:14301435. pyruvate carboxylase (Section 9.9), and PEP car-
Find the fermentation balance according boxykinase (Section 9.13). The latter enzyme usually
operates in the direction of PEP synthesis; however,
to both the O/R method and the available in some anaerobes (e.g., Bacteroides) it functions to
hydrogen method. What percentage of the synthesize oxaloacetate.
glucose carbon is recovered in end products?
6. Gest, H. 1983. Evolutionary roots of anoxygenic
5. Consider the following data for fermenta- photosynthetic energy conversion, pp. 215234.
tion products made by Selenomonas rumi- In: The Phototrophic Bacteria: Anaerobic Life in
the Light. Studies in Microbiology, Vol. 4. J. G.
nantium in pure culture and in coculture Ormerod (Ed.). Blackwell Scientic Publications,
with Methanobacterium ruminantium (in Oxford.
moles per 100 moles of glucose). 7. Tasaki, M., Y. Kamagata, K. Nakamura, K.
Okamura, and K. Minami. 1993. Acetogenesis from
Proportion (mol/100 mol glucose) pyruvate by Desulfotomaculum thermobenzoicum
Product Selenomonas Selenomonas + and differences in pyruvate metabolism among three
sulfate-reducing bacteria in the absence of sulfate.
Methanobacterium
FEMS Microbiol. Lett. 106:259264.
8. Gas production is generally observed as a bubble
Lactate 156 68
in an inverted vial placed in the fermentation tube.
Acetate 46 99
The bubble is due to H2, since CO2 is very soluble in
Propionate 27 20
water.
Formate 4 0
Methane 0 51 9. Jones, D. T., and D. R. Woods. 1986. Acetone
CO2 42 48 butanol fermentation revisited. Microbiol. Rev.
50:484524.
Source: Chen, M., and M. J. Wolin. 1977. Inuence
of CH4 production by Methanobacterium ruminan- 10. Hamilton, W. A. 1988. Energy transduction in
tium on the fermentation of glucose and lactate by anaerobic bacteria, pp. 83149. In: Bacterial Energy
Selenomonas ruminantium. Appl. Environ. Microbiol. Transduction. C. Anthony (Ed.). Academic Press,
34:756759. New York.