Degradation of Glucose-6-Phosphateto Pyruvate 15

coupledto a conversionof the substrate (glucose)intoa derivative

of the
substrate (glucose-6-phosphate),
are calledtranslocation
processes.

I1. Degradation of
Glucose-6-Phosphate
to Pyruvateviathe
Embden-Meyerhof-Parnas
(EMP) Pathway
The EMP pathway was firstdiscoveredin muscle tissues. Itisthe most
commonly used sequence of reactionsfor the conversionof
glucose-6-
phosphate intopyruvate and occursinanimals,plants,and many bacteria.
E. colicontainshigh activities
of the necessary enzymes (Table2.1).
the firsttwo reactions
In
catalyzedby the enzymes glucosephosphate isom-
erase and
phosphofructokinase,glucose-6-phosphate is converted into
D-fructose-1,6-bisphosphate,the characteristicintermediateof thispath-
way (Fig. 2.2).Fructose bisphosphatealdolase
splitsfructose-1,6-bis-
phosphate into two C3
fragments-dihydroxyacetonephosphate and
D-glyceraldehyde-3-phosphate. Both compounds are in
each other due to the equilibriumwith
presence of the enzyme triosephosphate isomerase.
The equilibriumconstant of the isomerase
reactionfavorsthe dihydroxy-
acetonephosphatebut the enzyme triosephosphate isomeraseisso active
(compared to other enzymes of the EMP
pathway) that immediate
conversion of the ketose derivativeinto the
aldose isomer occurs.

Table 2.1.Activity of the enzymes of the

pathway in cellextractsof E. coli Embden-Meyerhof-Parnas

average specificactivity
enzyme
(units/mgprotein)

glucose phosphate isomerase 1

phosphofructokinase 0.3
fructosebisphosphate aldolase
0.1
triosephosphate isomerase
4
glyceraldehyde-3-phosphatedehydrogenase 1.2
3-phosphoglyceratekinase 2.2
phosphoglycerate mutase
enolase
pyruvate kinase 0.7

One enzymeunit is definedas that amount of

enzyme that catalyzesthe
of 1
conversion umol of substrate(s)
totheproduct(s)in 1 min at 25°C.
Data obtainedas personalcommunicationfrom D.G.
Fraenkel(Boston,MA,
U.S.A.).
 ATP duringAerobic Growth on
coliSynthesizes
2: How Escherichia
16 0se

CH,OPO,H

C#0 H,0,POH,C CH,OPO,H

HO-C-H
H-C-OH
H OH
H-C-OH
OH
CH,OPO,H

Fischerprojectionof the
Figure2.2.Structure of fructose-1,6-bisphosphate.
a:
of the furanose form.
open form.b: Haworth projection

Consequently, ifonly little

dihydroxyacetonephosphate isneeded by the
most of the Cg fragments originating
cells, from fructose-1,6-bisphosphate
can be furthermetabolized viaglyceraldehyde-3-phosphate.This iswhat

normally happens (Fig.2.3).

The oxidation of glyceraldehyde-3-phosphateto pyruvate isinitiated
by
two enzymes, which togetheraccomplish the oxidation of the aldehyde

group to a carboxylgroup. The firstof these is D-glyceraldehyde-3-

and the reaction
phosphate dehydrogenase. Itcontains bound NAD,
proceeds as shown in Fig.2.4.
The 1,3-bisphosphoglycerate formed is a mixed anhydride of D-3-
of
phosphoglycerateand phosphate,and the freeenergy of hydrolysis this
anhydride ishigherthan that forATP ADP + P,.Therefore,the
conversion of 1,3-bisphosphoglycerate
to 3-phosphoglyceratecan be cou-
pled to the phosphorylationof ADP to ATP. The enzyme catalyzingthis
reactionis3-phosphoglyceratekinase.This isone of the two sitesof the
EMP pathway that yieldATP by substrate-level phosphorylation:

D-1,3-bisphosphoglycerate ADP
+ kinase
phosphoglycerate

D-3-phosphoglycerate t ATP
Inthe next two steps 3-phosphoglycerateisconverted to phosphoenol-
pyruvate.Firstphosphoglyceratemutase transfersthe phosphorylgroup
from position
three to positiontwo of glycerate.The enzyme requires
2,3-bisphosphoglycerate as cofactor.Enolase (phosphopyruvatehydra
tase)removes water to yieldPEP, Thisisanother compound containinga
phosphoryl bond with a high free energy of hydrolysis,and during the
formationof pyruvate from PEP itistransferredto ADP to yieldATP.
The enzyme catalyzing thisreactionispyruvatekinase(secondsiteofATP
formation in the EMP pathway):

PEP + ADP Pyruvatc kinase

Pyruvate + ATP
Since PEP isrequiredforthe glucose transportsystem, only halfof itis
availableforthe pyruvate kinasereaction.
 Degradationof Glucose-6-Phosphate
to Pyruvate 17

CH,OH
AT ADP) CH,O
C0
HO-C-H HO-C-H 6-
fructose-1,
H--OH H-C-OH bisphosphate

H--OH H-C-OH
CH,o CH,o
fructose-6-phosphate

CH,OH
- HC-0
H-C0
H--OH
HO--H
dihydroxyacetone-
phosphate

CH,o
P+NAD-
S - H-C-OH
CH,O
NAD+P
glyceraldehyde-3-
phosphate

H--OH -NADH H
INADH+H
H--OH
CH,o COo coo
glucose-6-phosphate HC-OH 1,3-bisphospho
HC-OH glycerate
CH,O CH,O
ADP) ADP
ATR ATD
COOH COOH
glucose 3-phospho-
HC-OH HC-OH giycerate

CH,O CH,O

COOH CoOH
2-phospho
Hc-o HC-o glycerate
CH,OH CH,OH

H,O H,0
COOH

--o
H
-o
CoOH

CH
phosphoenol-
Pyruvate

ADP
10
ATP
ÇoOH CoOH
Pyruvate

H CH

Figure2.3.Uptake ofglucoseand breakdown ofglucose-6-phosphateto2 pyruvate

viathe Embden-Meyerhof-Parnaspathway. 1,PEP:glucosephosphotransferase;
2,glucosephosphate isomerase;3,phosphofructokinase;4, fructose
bisphosphate
aldolase;5,triosephosphate isomerase;6,glyceraldehyde-3-phosphatedehydro-
9,
genase; 7, 3-phosphoglyceratekinase;8, phosphoglyceratemutase; enolase
10, pyruvatekinase
18 coliSynthesizes
2:How Escherichia ATP duringAcrobicGrowth on
Glucosu

NAD NADH +
H
H-C-0
E SH H-C-O -C-0
CH,O H-C-OH
CHO
NADH+ H NAD

ES-C-o NAD -C-0+NADH+ H

H-C-OH --OH
CHO CH,o

NAD NAD
O-C-0-C
ES-C-O +P SH+ H-C-OH

H-C-OH CH,O
CH,O
Figure2.4.The conversion of glyceraldehyde-3-phosphate
to 1,3-bisphospho
glycerate.In the initialreactionthe aldehyde is oxidizedto a thioester
and the
reducingpower is transferredto the enzyme-bound NAD". An exchange reaction
then takes place with solubleNAD. Finally, the acylgroup on the enzyme is
transferredto inorganicphosphate to yieldthe 1,3-bisphosphate.

The sum of the reactionsdiscussedthusfaris:

glucose + PEP + pyruvate

glucose-6-phosphate
glucose-6-phosphate+ ADP +2NAD +2P
2PEP+ ATP +2NADH+ 2H
PEP +ADP- Pyruvate + ATP
glucose+ 2ADP + 2NAD + 2P
2 pyruvate+ 2ATP +2NADH + 2H
NADH dehydrogenase re
isformed in the glyceraldehyde-3-phosphate
actionand ATP by 3-phosphoglyceratekinaseand pyruvate kinase.One
ATP isconsumed in the phosphofructokinasereaction.

Il1.OxidativeDecarboxylationof Pyruvate
to Acetyl-Coenzyme A
As in most aerobicmicroorganismsthe formationof acetyl-coenzyme A
(acetyl-CoA) from pyruvate in E. coliis catalyzed by the pyruvate
dehydrogenase complex.This complex consists
of threeenzymes:24 n
cules each of pyruvate dehydrogenase (E,)and dihydrolipoate t
acetylase (E,)and 12 moleculesof dihydrolipoate dehydrogenase (E3)
The E molecules form the core of the complex, and the and E E
moleculesare bound to the outside of it.E, containsthiaminepyrophos
 of Pyruvate to Acetyl-Coenzyme A
OxidativeDecarboxylation 19

phate (TPP),and the firststep in the oxidativedecarboxylation

is the
additionof pyruvate to C-2 of the thiazoliumring of TPP to form
lactyl-TPP-E.
NH CH
(CH;)---enzyme
-CH2
H,C
H,C-C-c
o

NH2 CH
(CH,)-P--enzyme
N -CH2

H,C H,C-COH

O
The furtherreactionsactingupon the lactylresidueare summarized in
Fig.2.5.Decarboxylationyieldshydroxyethyl-TPP-E. The hydroxyethyl
from TPP to the lipoategroup of E2.Concom-
moiety isthen transferred
itantlythe disulfide
bond of lipoateisreduced.The acetyl group thus

TPP+CH,-CO-COOH TPP-CH-CH, +CO

OH

E,FAD FAD

E,

BC S-CO-CH
CoASH
Pa SHCH-CO-SCOA
EFAD FAD
TPP TPP

NAD
NADH+ H'

E FADH FAD
sum:CH-CO-COOH+CoA+ NAD CH-CO-CoA +CO +NADH+ H
Figure 2.5.Reactions catalyzedby the pyruvate dehydrogenase complex. E
Pyruvate dehydrogenase; E2, dihydrolipoate Es,dihydrolipoate
transacetylase;
dehydrogenase,TPP,thiaminepyrophosphate;thedisulfidecompound linkedtoEz
is the oxidizedform of
lipoate
20 9 How coliSynthesizesATP duringAerobic
Escherichia Growth on Glue
lucose

formed is then released as acetyl-COA and under

catalysisof E.th
form of lipoateisoxidizedby NAD
sulfhydryl to the disulfide
form.Th
enzyme complex is then ready for the oxidationof another molecule oe
pyruvate to acetyl-CoA.
The combined action of the enzymes of the EMP
pathway and the
pyruvate dehydrogenase complex leads to the degradationof glucoseto
CO and acetyl-CoA according to the equation:
glucose+2ADP +4NAD + 2P,+2CoA
2acetyl-CoA+2CO2 + 2ATP + 4NADH
+4H

W. Oxidationof Acetyl-CoAvia the

Acid Cycle
Tricarboxylic
Thiscyclewas discoveredby Egglestonand Krebs in animal tissues.Itis
oftenreferred to as the Krebs or citricacid cycle.That itispresentin
E. colihas been demonstrated with enzymatic methods and withexperi
ments using radioactive substrates.The cyclecarriesout the oxidation of
the acetylmoietyof acetyl-CoA to CO, withtransfer of the reducingequi
valentsto NAD, NADP, and FAD. Acetyl-CoAenters the cycleby
the citratesynthase reactionin which oxaloacetateand acetyl-CoAare
condensed to give citrate. In the subsequent reactionsof the cycle the
Ca-acceptor for the next molecule of acetyl-CoA, oxaloacetate, israther
elegantlyregenerated.
As shown in Fig.2.6 citrateis firstisomerizedto isocitrate.Thisis
accomplished by a dehydration to enzyme-bound cis-aconitate, which
is
subsequently hydrated to give isocitrate.
Next, isocitrateundergoes
oxidationto oxalosuccinate,
which isdecarboxylatedby the same enzyme,
dehydrogenase,to yielda-oxoglutarate.
isocitrate Likethe corresponding
enzyme of most otherbacteriathe isocitratedehydrogenase of E. coli1s
NADP-specific. Oxidationof a-oxoglutarateto succinyl-CoAiscatalyzed
ot
by an enzyme complex analogousto the one involvedinthe oxidation
pyruvate to acetyl-CoA.Italsocontainsthree enzyme species,one cata-
lyzingthe decarboxylationof TPP-bound a-oxoglutarate, one accepting
the succinyl
moiety and succinyl-CoA,
releasing and one withdihydrolip0ate
dehydrogenase activity. The dihydrolipoatedehydrogenase is identical
to the correspondingcomponent of the pyruvate dehydrogenase complex
and ithas been shown that E. colicontainsa singlegene locus for this
enzyme. Thus,newly synthesized dihydrolipoate willcom
dehydrogenase
bine with two enzyme components to yieldeitherthe pyruvate o
a-Oxoglutarate dehydrogenase complex.
Inthe next step the energy ofthe thioester
bond ofsuccinyl-CoAIS U
to synthesize ATP from ADP and inorganicphosphate.This isanotnc
reactionin which ATP isgenerated by substrate-level phosphorylatio
Oxidationof Acetyl-CoAvia the Tricarboxylic
Acid Cycle 21

NADP
HO-CH-COOH
NADPH+H
H,O H-COOH
CH,-CoOH OC-COOH
CH-COOH isocitrate
Oxalo
CH-COOH
C-COOH cis-aconitate
Succinate CH-COOH
H,OCH-COOH
ÇH-COOH OC-CooDH
citrate o-Oxoglutarate CH2
HO-C-COOH
CH,-COOH
CoASH
CH-COOH
CoASH C0
NAD'
CCOSo H,0
NADH +
ço-coOH oxaloace tate CO-SCoA
H-COOH succiny1-CoA CH
NADH H'F CH,-COOH

NAD'
10 L-malate
succinatte ADP
HO-CH-COOH ATD
CH-COOH CH-CoOH
fumarate COASH
9 HO CH- COOH
CH-COOH FAD
HOOC-CH
FADH,
acid cycle.1,citrate
Figure2.6.Oxidation of acetyl-CoA via the tricarboxylic
synthase;2 and 3,cis-aconitate
hydratase; 4 and 5, isocitrate
dehydrogenase;6,
succinate
a-oxoglutaratedehydrogenase complex; 7, succinatethiokinase;8,
dehydrogenase;9,fumarase; 10,malate dehydrogenase.
succinatee

succinyl-CoA + P+ ADP thiokinase

succinate+ ATP + CoA
The correspondingmammalian enzyme phosphorylatesGDP or IDP
but not ADP. Sucinateisoxidizedto fumarate by succinate dehydro
associated with the cytoplasmic
residesin particles
genase. The enzyme
membrane and transfers electronsfrom succinateto bound FAD. As
willbe discussedlaterthe electronsare then channeled from FAD into
the respiratory chain.That NAD is not used as electronacceptorin
this oxidation reaction is because the fumarate/succinate system
(E6= +0.03 V) has a more positiveoxidation-reduction potentialas
compared to that ofNAD /NADH + (E = -0.32 V).Thus,succi
H
nate isa very weak reductantforNAD . Protein-boundFAD with E6 of

approximately-0.06V ismuch more suitable.

Two additionalenzymes are necessary to generate oxaloacetate. First
fumarase hydrates fumarate to L-malate,then L-malate dehydrogenas
oxidizesL-malate to with
oxaloacetate NAD* as H-acceptor.
acidcycle
With the oxidationof two acetyl-CoA viathe tricarboxylic
whichtheoreticallyis
glucoseiscompletelyoxidizedtoCO2. The hydrogen
 2:How SynthesizesATP duringAerobicGrow
Escherichiacoli on
22 ucose

in the form of reduced coenzvmes

availableinthisoxidationisconserved
is usually calculated by oxidizing
The number of availablehydrogens the
with water; for glucose it is 24(H):
substrate(on paper)
to CO
CH20, +6H,0 24(H) +
6CO
availablehydrogen
glucose

+ 2NADP" +2FAD + 4ADP +

4P,
glucose +8NAD
8NADH + 2NADPH + 10H+2FADH, + 4ATP +6CO.

24(H)
soon would come
Itisclearthat the process of glucoseoxidation to a
to regenerate the oxidizedforms of the
standstillifthere were not reactions
the principal H-acceptorduring
coenzymes.As inotheraerobicorganisms
apparatus used
the to react the
aerobicgrowth of E. coliis oxygen and
is the respiratory
reduced forms of the coenzymes with oxygen chain
be mentioned that E. coli is a
However, in thisconnection it must
of the
anaerobe and that even under aerobic conditions part
a
facultative
viafermentative pathways not involvingoxygen.For
glucose iscatabolized
the sake of simplicity the simultaneouslyoccurringfermentativemetabo-
in a later
lism of E. coliwillbe neglectedhere and willbe discussed
chapter.

V. The Formationof ATP in the

Chain
Respiratory

A. Oxidation-reduction
potential
(OR) reactionmay be writtenas follows:
An oxidation-reduction
Ared Aox tnelectrons
Box +n electrons F Bred

Ifan equimolarsolutionof Aped/Aox isaddedto an equimolar solution

of Bred/Bx,the direction of any reactiondepends on the tendency of the
Ared/Aox 5ystem to donate electronstothe Bped/Bo,system and vice versa.
A quantitativemeasure of this"tendency"in OR systems istheirredox

potential.
the hydrogen electrode-a solution
To measure redox potentials con-
taining H of unit activity(pH 0) and an inertmetal electrodein
equilibriumwith H2 at atm-is normallyused as referenceelectrode.its
1
oxidation-reduction taken to be zero (Eo = 0 V).
potentialisarbitrarily
Spontaneouslyorinthe presence ofthe appropriatecatalysts, OR systeins
a
with negative redox reduce
potentials H to hydrogen. OR systems with
E
positive are reduced by H2. The dependency of the oxidation-reduction
The Formationof ATP in the Respiratory
Chain
23

potentialon the concentrationof the components of the OR system is

expressedby the Nernst equation:

[ox]
E -Eo+
F"[red]
(R,gas constant;T,absolutetemperature;n, number of electrons;F,
Faraday constant).
In allreactionsinvolvingprotons the standard oxidation-reduction
potentialrefersto pH = 0. Inthat most biological
reactionsproceed at pH
values near 7 itis more practicalto calculatethe standard
oxidation
reductionpotentialof biological when
systems the pH is
7.
At pH 7 and 30°C the potentialof the hydrogen electrodebecomes
-0.42 V:

E
(pH 7)
E F
(pH-0)
+
n
In10-7
ln10-7

The
E6=0+
E valuesof NAD
1
8.314 x
x96494
and
S052.303x -7-0.42
V

O, are -0.32and +0.82 V,respectively,

and
the differencebetween them isthe ofthe respiratory
chain.
potential
span

B. Components of the respiratory

chain
The
major components of the respiratory
chain are proteinsbearing
with
prosthetic groups oxidation-reduction potentialslyingbetween those
of NAD and oxygen. In the mitochondrialmembrane of eukaryotic
and membrane of bacteria, these proteinsare
organisms the cytoplasmic
arranged in a
such way that the reducingpower of NADH can flow to
viacarriers o f increasingoxidation-reduction potentials as ifover
Oxygen
cascades. However,the compositionof the E. colirespiratory chainisnot
identicalto that of mitochondria (Fig.2.7).As is indicatedby the
rectangles the mitochondrial chaincontainsfourcomplexes,which can be
isolated 1
as such.Complex isthe NADH dehydrogenase;itcontainsflavin
mononucleotide (FMN) and iron-sulfur proteinsand transfershydrogen
from NADH to coenzyme Q. Succinate
dehydrogenase (complex 2) also
feeds hydrogen into the chain at the coenzyme Q level. This
respiratory
enzyme is a flavinadenine dinucleotide
(FAD)-containing protein.
Cytochrome c is then reduced by coenzyme Q viacomplex 3 and finally,
complex 4 catalyzes the transferof reducing power to oxygen.
The compositionof the E. colirespiratory chainisdifferent from that of
mitochondria;cytochrome c is not involved, and, most noteworthy, the
E. colichain is branched. Incells under aerobicconditions
growing fully
tows
via coenzyme O, cytochrome bss
and
reducingpower preferentially
cytochrome o to oxygen. Oxygen-limitedcellsemploy coenzyme Q or
menaquinone and cytochromesbssg,a,and d as carriers.
 ATP duringAerobicGrowth on
coli
2:How Escherichia Synthesizes Glucose

ot carriers
The structuralformulasof the tourtYpes present in resnirs

inFigs.2.8to2.11.Flavoproteins, coenzyme O.and

torychainsare given reductiontwo hydrogensaare
are hydrogen carriers.
During
menaquinone
NADH+H NAD
NADH+H NAD

flavoprotein
lavoprotein FeS-protein
succinate fumarate
FeS-protein

succinate coenzyme Q
coenzyme Q dehydrogenase (menaquinone)

cytb
cyte
cytbss6 cytbss

cytc
cyto cyta,d

cyt a
cyta

H0 H20

H,0c motsigassert 020000

mitochondria E.coli

Figure2.7. chainsofmitochondria and of E. coli.

Components of the respiratory
growing under real aerobic
The E. colichain isbranched; a dominates incells
b dominates inoxygen-limited
conditions, cells. iron-sulfur
FeS-protein, protein;
cyt,cytochrome.

CH NH
CH-
CH2
CHOH
CHOH
CHOH
CHOPO H2

Figure 2.8.Flavinmononucleotide (FMN), the prostheticgroup of the NADn

dehydrogenase of the respiratory
chain.Circlesindicatewhere reductiontakes
place. Many other enzymes including succinatedehydrogenase contain flavin
adeninedinucleotide (FAD).The oxidation-reduction offlavoproteins
potential
with the
not identical ofFMN and FAD (E=-0.19 and-0.22
potentials
Due to interaction
respectively). of theproteinwith its
groups E can
De
prosthetic
eithermore negativeor more positive.
 CHO -CH3
CHO
CH,CHsCH;H
tcH
CHs

CH

CH H H

CH
Circlesindicate
Figure2.9.Coenzyme Q (ubiquinone)(a)and menaquinone (b).
where reductiontakes place.n variesfrom 4 to 10; in E. coli,n = 8 for both

quinones.

CH CH CH Chi CH CH
CH CH
CH CH CH CH
HO-CH CH HC=CH CH

H,C -CH=CH2 H,C -CH=CH

-CH H,C -CHs

o-C

CH CH CH2 CH
HOOC-H CH-COOH HOOC-CH, CH-COOH

cys

H,C-CH CH
cys 2 ueo HC-OH CH

HC -CH-CH H,C- -CH=C

H,C- CH3 H,C7 -CH

H
CH CH2 H CH CH
HOOC-CH CH,-COOH HOOC-CH CH-CO0OH

Figure2.10.The prosthetic groups of the cytochrome types.The prostheticgroup

of
the a-type cytochromes containa formylgroup as sidechain (heme a).The
of
prostheticgroup the b-typecytochromes isheme b.Incytochromes of thec-type
the to the proteinviacysteineresidues.The
prostheticgroup iscovalentlylinked
group of the d-typecytochromes isa derivative of dihydroporphyrine
prosthetic
The structuresof Ri, R2, and R, are unknown.
 coliSynthesizesATP duringAerobicGrowth
2:How Escherichia o
26

cysteine-S S-cysteine

S-cysteine
cysteine-S

peptide chain

[2Fe:2S]
center

protein.Iron is bound
The 2Fe:2S] center of an iron-sulfur to
Figure2.11.
of the peptidechain and to sulfide.
cysteineresidues

transferredto one carriermolecule:

2H
carrier+ carrier-H2
proteinsare electroncarriers. Four classes
Cytochromes and iron-sulfur
of cytochromes are distinguished: cytochromes a, b, c, and d. The
structural differences between them are indicatedin Fig.2.10. These
differenceshave an effecton the redoxpotential of the cytochromes which,
however, salsoinfluenced by the electronaffinity of the proteinligands
withthe centralFe atom. The redoxpotential can vary between
interacting
E-0.2 V (low-potential cytochromes) and E6= +0.4 V
(cytochrome
ag).Cytochrome o does not represent a fifth classof cytochromes;itrather
isa b-type cytochrome with oxidaseactivity. It is oxidized by
O like
cytochrome ag and ispresent inthe E. colirespiratory chainunder aerobic
conditions (see Fig.2.7).Inthe iron-sulfurproteinsthe ironisnot bound
to a heme group.Itisbound to the sulfhydryl groups ofcysteineresiduesof
the protein. Inaddition,ironatoms are linkedto one another by sulfur
bridges so that centers are formed. Most of these are of the
iron-sulfur
[2Fe:2S] type (Fig.
orof
2.11) the [4Fe:4S]type (see Chapter 8).
Duringreductionof a cytochrome oran iron-sulfur proteinone electronis
transferred tothecentralironofthe cytochrome and toone ofthe irons ofthe
FeS centerof the iron-sulfur
protein, respectively:
carrier Fe3+H carrierFe2 + H
Thus,ifelectroncarriersare reduced by hydrogen carriers,protons are
released. Conversely,the reduction of
hydrogen carriersby electron
carriersrequiresprotons.This isimportant, because hydrogen and elec
arranged in alternatingsequence ina membrane may causc
tron carriers
proton translocations;
protons releasedmay be excreted at one side the
oft
114 S:MetabolicDiversityof Aerobie
Heterotropl
ophs

It thatribosomescarrying
isalsopossible the leader
peptide and the vet
incomplete
polypeptideassociatewith the membrane at sites
where
dockingproteinisinsertedintothe membrane. The
polypeptideis then
completedwhileitisalreadypulledthrough the membrane
(Fig.5.8b).
Assembly of proteinsintomembranes or proteinexport
requiresthat
the membrane is in an energizedstate.These
processes are proton
motive-force-dependent. For the insertion
of some proteins, the simul.
taneous insertionof phospholipidmoleculesis necessary,
Transportof
proteinsinto membranes is a very complex prOcess, and there are still
many questionsto be answered.

ED Paihud
I1.
The Entner-Doudoroff
Pathway
Besidesthe
Embden-Meyerhof-Parnaspathway, there is a second im-
portantpathway used forcarbohydratebreakdown, which is
widely dis-
tributedamong bacteria.It was firstdiscoveredby Entner and Doudo-
roffin Pseudomonas
saccharophila.As in the pentose phosphate cycle,
glucose-6-phosphateisfirstdehydrogenatedto yield6-phosphogluconate
(Fig.5.9).Thisisconvertedby a
dehydratase and an aldolasereactioninto
1 molecule of and 1 molecule of
3-phosphoglyceraldehyde pyruvate. The
3-phosphoglyceraldehydecan then be oxidizedto pyruvate by the enzymes
of the
Embden-Meyerhof-Parnas pathway.
What are the differencesbetween the
Embden-Meyerhof-Parnas
(EMP)and theEntner-Doudoroff (ED)pathways and how isitpossibleto
differentiate
between them? The key
enzymes of the EMP path
way-meaning the enzymes common only to this 1-
pathwayare and
6-phosphofructokinase,respectively.All the otherenzymes of the EMP
pathway may be partofothermetabolicsequences as well,Fru-P2aldolase,
forinstance,in
gluconeogenesisor inthe pentose phosphate cycle.The key
enzymes of the ED pathway are
6-phosphogluconate dehydratase and
2-keto-3-deoxy-6-phosphogluconate(KDPG) aldolase.Thus, these path

COOH
HC-O COOH C-0
NADPH+ H COOH
HC-OH CH
HC-OH
HO- CH NADP HO Pyruvate
HO CH
HC-OH CH
HC-OH OH
HC-OH Ho HC-OH
HÇ
HC-O
HC OH HC-OH
CH,o CH,o- CH,o CH,0-
gucose-6-PD 6--guconate 2-keto-3-deoxy- 3--slycerak
6-(P)-gluconate dehyde
(KDPG)
Figure5.9.Reactionsof the Entner-Doudoroff
pathway. 1,Glucose-6-phosphate
dehydrogenase; 2, 6-phosphogluconatedehydratase;3,KDPG aldolase.
 The Entner-Doudoroff
Pathway
115

1
CH
2HC-OH 2C-0 2 COSCoA
3 HO
CH EMP 3 COO
4
HC-ON 4 COOH
3CO
4 CO,
HC-ON sC-O 5
COSCoA
CH,OR 6 CH 6 CHy

HC-Oo 1
COOH ICO
2 HC-ON 2 C-O 2 COSCoA
3 HO-CH
4
ED 3 CH 3 CH
HC-OH 4 COOH
4 CO
HC-OH sCO 5 COSCoA
CH,OH 6 CH 6 CH
Figure5.10.Originof the CO-carbon formed in the
reactionafterdegradationof pyruvate dehydrogenase
glucoseby eitherinthe
(EMP) or the Entner-Doudoroff(ED)pathway. Embden-Meyerhof-Parnas

ways can be distinguishedin the

followingway: cellsof a particular
bacterium are grown on glucose,cellextractsare
prepared,and the key
enzymes of the two pathways are assayed. If levelsofthe dehydratase
high
and KDPG aldolaseare and
present phosphofructokinases are not detect
able,itislikely that the ED
pathway isinvolvedin glucosedegradation.
A second method, radiorespirometry, alsobe used to
might determine
whether the EMP or ED pathway existsina
for thisisillustrated
particular organism.The basis
in Fig.5.10.It is obvious that both
pathways yield
"different"pyruvate from the first3 carbon atoms of
glucose. Inone case
(EMP) the carboxylgroup originates from carbon 3 of
glucose,inthe other
(ED) from carbon 1 of glucose.During aerobicoxidationof
first glucose the
CO2 appearingcomes from the oxidativedecarboxylationof
it thus pyruvate;
originatesfrom glucosecarbon atoms 3 and 4 (EMP) or 1 and 4
(ED). This differenceis detectableby
radiorespirometric methods.For
instance,glucose labeledwith "C in
C, C3, or Ca is added to cell
suspensions of Bacillussubtilis and Xanthomonas
The CO
evolvediscollectedevery hour and its phaseoli.
radioactivityisdetermined.Itis
evidentfrom Fig.5.1l that B. subtilis releases fasterfromC
radioactivity
and C3 of glucoseand X. from C and Ca;thus,the first
phaseoli bacterium
employs the Embden-Meyerhof-Parnas and the second
the Entner-
Doudoroffpathway.
Which of these two
pathways ismainly involvedin the breakdown of
hexosesby variousbacteriaissummarized in
Table5.2.As can be seen,the
Entner-Doudoroff pathway is fairlywidespread,
particularlyamong
Gram-negative bacteria,
whileit isnot verycommon in
of anaerobes.Inview
the factthat anaerobes do not
have a chain and gain their
ATP usuallyby substrate-level respiratory
economical for them; the phosphorylation,
the EMP is
pathway more
EMP pathway yields2ATP in the conversion
 of Aerobic
5: MetabolicDiversity
116 Heterotrophs

subtilis Xanthomonas phaseoli

Bacillus

A
Figure5.11.
time

Evolutionofradioactive
4 by cellsuspensionsofB. subtilis

pp. 30-63 (1961)

(1967).]
and X. phaseoli.
Metabolic Pathways in Microorganisms.
time

CO2 from glucoselabeledinpositions1,3,or

[Data from V. H. Cheldelin,.
John Wiley and Sons,Inc.,New York,
and A. C. Zagalloand C. H. Wang. J.Bacteriol, 93,970-975

of 1 glucose into2 pyruvates;the ED pathway yieldsonly 1ATP per 2

Pyruvates.
The ED pathway isalsoimportantwhen compounds such as gluconate
mannonate, or hexuronates serve as substrates.If E. coliistransferred
from glucosemedium
a toa gluconatemedium, the followingenzymes are
formed;gluconokinase,which phosphorylatesgluconateinposition 6,and
the two key enzymes of the Entner-Doudoroff pathway. Thus, in E. coli
glucose and other hexoses are degraded via the EMP pathway, but
gluconateand relatedcompounds via the ED pathway (Fig.5.12).
Clearly,the EMP pathway isnotveryusefulforgluconatedegradation.
A reaction sequence leading directlyfrom 6-phosphogluconate to

Table5.2.Pathways involvedinsugardegradationby bacteria

microorganism EMP ED
Arthrobacterspecies
Azotobacterchroococcum
Alcaligenes
eutrophu
Bacillus B.cereus + otherspecies
subtilis,
coli+ otherenterobacteria
Escherichia
Pseudomonas saccharophila,
P.fluorescens+ otherspecies
Rhizobiumjaponicum + otherspecies
Thiobacillus
intermedius,
Th.ferrooxidans
Xanthomonas phaseoli+ other
species
 The Entner-DoudoroffPathway
117

glucose gluconate

PEP ATP
ruvate gluconokinase
Py
ADP
glucose-6-P 6-phosphogluconate

EMP pathway ED pathway

Figure5.12.Degradation of
glucose and gluconate by E.coli.

glucose-6-phosphateisnot known, and many bacteria

utilizegluconate,
mannonate, glucuronate,and related
Itshould be mentioned in compounds via the ED pathway.
thisconnectionthata number
nads also of pseudomo-
degrade glucoseviagluconate.P.
P. putida contain a aeruginosa, P. and
fluorescens,
glucose dehydrogenase, which converts
gluconate.The lattercan be glucose to
alternatively
phosphorylated to
gluconate or furtheroxidizedto 6-phospho0
2-ketogluconate,which is
phosphorylated and reduced to yieldalso subsequently
Glucose and 6-phosphogluconate (Fig.5.13).
gluconate dehydrogenases are
electronsdirectlyintothe membrane-bound and feed
respiratorychain. These enzymes allow the
extracellularconversion of
glucoseintocompounds thatare less
by othermicrobes than utilizable
glucose.Thus,these
monads, which can enzymes benefit the pseudo-
transportand degrade glucose,
ketogluconate. Fructoseis metabolized gluconate, and 2-
ventionalEntner-Doudoroff by these organisms viathe con-
route.

gucose 10 gluconate H,0

2-ketogluconate

glucose
membrane gluconate
dehydrogenase
dehydrogenase

glucose
gluconate
cytoplasm
ATP 2-ketogluconate
ATP ATP
ADP NAD' NADH+ H ADP NADP NADPH+H ADP
gucose-6-D 6-D-guconate Z-2-keto-6-
(P)-suconate
ED pathway
Figure5.13. Extracellular
oxidation ofglucoseby
conversion of pseudomonads and intracellular
glucose,gluconate,a nd
1, Hexokinase; into
2-ketogluconate
2, gluconate
kinase;3, 6-phosphogluconate.
phosphate 2-ketogluconatekinase;4, glucose-6-
dehydrogenase; 5,
Whiting,M. Midgley,and E. A. 2-keto-6-phosphogluconate reductase. P. H.
Dawes, J. Gen. Microbiol,
92,304-310(1976).1
I.AlcoholFermentation
A. Ethanolfermentation
by yeasts
Alcoholfermentation isthe domain of yeasts,notablyof Saccharomyces
species,and most of the ethanolformed in natureand produced by the
fermentation comes from the anaerobicbreakdown of glucoseand
industry
otherhexoses by these organisms.Gay-Lussachad alreadyestablished in
1815 that hexoses are convertedintoethanoland CO, accordingto the
 + +

6298
++t
 212 8:Bacterial
Fermentations

followingequation:

CgHi20% 2CH,OH +2CO

Figure8.1summarizesthe alcoholfermentationas
carriedout by
Itisapparent that yeasts yeasts,
employ the
forglucosedegradation. Embden-Meyerhof-Parnaspathway
Thus,2 mol of pyruvate are formed
from 1 mol of
glucose.Pyruvate,however,isnot convertedto
metabolism, but is decarboxylatedto acetyl-CoAas in aerobic
acetaldehyde.The enzyme that
catalyzesthisreactionispyruvate
decarboxylase; itcan be
key enzyme of alcohol fermentation. regarded as the
bound thiamine Pyruvate decarboxylase
contains
pyrophosphate;infact,thefunctionof thiamine
phate was first studied using this pyrophos-
enzyme. As in the pyruvate
drogenase reaction, dehy-
hydroxyethyl-thiamine pyrophosphate enzyme isan
intermediate.The
hydroxyethylgroup, however,is not oxidized
releasedas free but is
acetaldehyde.The latteristhen reduced to ethanol
alcoholdehydrogenase. by
One importantfeatureof
fermentationsisapparentfrom
even hydrogen balance.Since Fig.8.1:an
there is no external
H-acceptorsuch as
glucose

2ATP
2ADP
fructose-1,
6-bisphosphate

2 ethanol 2
glyceraldehyde-3-(P
2NAD
-2P

2 acetaldehyde
2NADH+ 2H
2 1,3-bisphosphoglycerate

2C0 2ADP)
2 pyruvate QATP)
2 3-phosphoglycerate
2ATP
(2ADP
2
phosphoenolpyruvate 2 2-phosphoglycerate

2H,0

Figure8.1.Fermentation of glucose to ethanol and CO by

yeasts.1,Initial
enzymes of the Embden-Meyerhof-Parnas
pathway; 2, glyceraldehyde-3
phosphate dehydrogenase; 3, 3-phosphoglyceratekinase;4,
phosphoglycerate
mutase; 5, enolase;6, pyruvate kinase;7,
pyruvate decarboxylase;8, alcoho
dehydrogenase.
AlcoholFermentation 213

oxygen, NADH-producingand NADH-consuming reactionshave to be

balanced out. This also implies that NADH formation is avoided by
anaerobes ifpossible.
Therefore,they do not oxidizeacetyl-CoAviathe
tricarboxylic cycle.The cycle is usuallyinterruptedbetween a
acid
Oxoglutarateand succinyl-CoA be formed from
so thatglutamate can still
Oxaloacetateand acetyl-CoAviacitrate

NH2 HC
(CH)-P--enzyme
CH

H,C
H
H,C-C-o
H
hydroxyethyl-thiaminepyrophosphate enzyme

B. The Pasteureffect
The net ATP yieldof the alcoholfermentationis2 mol ATP/molglucose
(Fig.8.1)much lowerthan the ATP yieldof aerobicmetabolism. Yeast
cellsrespond to thislarge difference. When they are transferredfrom
aerobic to anaerobicconditionsthey increasethe rate of glucose break-
down by a factorof3 to 4;a change from anaerobictoaerobicmetabolism
is accompanied by a reductionof thisrate and a stoppage of alcohol
formation.This phenomenon iscalledthe Pasteureffect.Apparently, the
is inthe cell'senergy charge under
Pasteur effect the resultof differences
aerobic and anaerobic conditions.In the presence of oxygen the respira-
torychainand thesitesof substrate-levelphosphorylationinthe glycolytic
pathway compete forADP. Furthermore, phosphofructokinaseactivityis
the of and citrate.Under anaerobicconditionsthe
controlled by level ATP
phosphofructokinase increasesbecause it is activatedby ADP
activityof
and AMP; also, more ADP is availablefor the enzymes
catalyzing
All thisallows a greatersub-
substrate-level phosphorylationreactions.
strate flow through the glycolytic
pathway.

C. Alcoholfermentationby bacteria
Itshould be mentioned that yeasts are not truly anaerobic
facultatively
organisms.They grow only for some generationsunder these conditions
There are, however,some bacterialspeciesthat
carry out an alcohol
fermentationand grow very wellunder anaerobicconditions.
Zymomonas
mobilisisolatedfrom Mexican pulque and the
closelyrelatedZymomonas
anaerobicadegrade glucoseto pyruvate viathe Entner-Doudoroff
path
way; they containpyruvate decarboxylaseand form nearly2 mol each
ethanol and carbon dioxide from I mol
ot
a
glucose.Sarcinaventricult,
anaerobe capable of strict
growth under extremely acidicconditions,and
214 :Bacterial
Fermentations

anaerobic enterobacterium,ferment
Erwiniaamylovora,a facultatively
to ethanol and CO2 viathe
glucose Embden-Meyerhof-Parnas pathway
and the pyruvate decarboxylase and alcohol
dehydrogenase reactions.
Both organisms,however, form small quantitiesof other
products in
addition:acetate and molecular hydrogen (S. ventriculi)or lactate(E.
amylovora).In general,pyruvate decarboxylaseisrarein bacteria.Many
lacticacid bacteria,enterobacteria,and clostridia form considerable
amounts of ethanol but do not employ pyruvate
decarboxylaseforacetal-
dehyde synthesis.In these organisms acetyl-CoAfunctionsas ultimate
precursorof acetaldehyde;itis reduced by acetaldehyde dehydrogenase:
acetaldehyde

CH3-CO-SCoA + NADH + Ht dehydrogenase,

CH-CHO + CoASH + NAD

The investigation
of the alcoholfermentationhas been of inestimable
importance tor the development of biologicalsciences.In 1897 the
Buchnerbrothers discoveredthatan extractof macerated yeast
fermente
glucose to ethanol.This demonstrated that a complex
biological proce
could functionoutside the cell.
In1905 Harden and Young discoveredthe importance of
phosphate
esters in metabolism; they
identifieda hexose bisphosphate (fructose
1,6-bisphosphate)as an intermediatein sugar fermentation.Later, the
path of glucose breakdown and the functionof the coenzymes in this
process were unraveled by Neuberg, Embden, Meyerhof, Parnas, and
Warburg.
Itshould be mentioned here that ethanolisnow
produced on a large
scaleby fermentationforindustrial
purposes.Bargasse(fromsugar cane),
molasses(fromsugar beets), and cornstarchareused as substrates;
yeasts
and to a small extent Zymomonas mobilisare employed as organisms.
Processesare currentlybeing developed that allow ethanol
production
from varioussubstratesby thermophiles, such as Clostridium
thermohydro-
sulfuricum,Thermoanaerobium brockii, or Thermoanaerobacterethanoli-
Cus. These
organisms,however, do not contain pyruvate decarboxylase;
they form ethanol via acetyl-CoAand excrete in additionto ethanol
considerableamounts of acetate and lactate.

I1. LactateFermentation
Lactateisa very common end
product of bacterialfermentations. Some
genera-often referredto as lacticacidbacteria-form largeamounts of
thisproduct.These microorganismshave in common that
they are highly
saccharolytic and that they lack most anabolic
pathways. So they exhibit
very complex nutritional requirements,which are met by theirenviron-
ment such as plantmaterials, milk, and the intestinal
tractofanimals.Most
 Lactate Fermentation 215

lacticacid bacteriaare strictly

fermentativebut are aerotolerant.Some
streptococci, however, can use oxygen as H-acceptor and even form
cytochromes under certainconditions.
InTable 8.3 speciesof the genera Lactobacillus, Sporolactobacillus,
Streptococcus,Leuconostoc, Pediococcus,and Bifidobacteriumare listed.
Either of three pathways is employed by these microorganismsfor the
fermentationof carbohydratesto lactate.
The homofermentative pathway yields2 mol of lactate per mol of
glucose:

homofermentative pathway 2 lactate

glucose

pathway yields1 mol each

The heterofermentative of lactate,
ethanol,
and CO per mol of glucose:

heterofermentativepathway
lactate+ ethanol + CO2
glucose

of
acidbacteriaand configuration
lactic
Table8.3.Homo- and heterofermentative
hacticacid produced

configuration
homofermentative heterofermentative of lacticacid
genera and species

Lactobacillus
L. delbrueckii D-)
L. lactis D(-)
L. bulgaricus D-)
L (+)
L. casei DL
L. plantarum DL
L. curvatus DL
L. brevis DL
L. fermentum

Sporolactobacillus
S. inulinus
D(-)
Streptococcus L(+)
S. faecalis
S. cremoris
L(+)
L(+)
S. lactis

Leuconostoc
L. mesenteroides
L. dextranicum D(-)
Pediococcus
DL
P.dumnosus
Bifidobacterium L(+)
B. bifidum
 8: Bacterial
Fermentations
216

acetate and lactatein a ratioof 3 to 2:

The bifidum pathway yields

2 glucose biñdum pathway 3 acetate +2 lactate

followedcompletelyby the lactic

These equationsare,of course,not
the yieldisfrequentiy1.8
acidbacteriabut in homofermenting organisms
bacteriaproduce 0.8 mol
mol lactate/molglucose.The heterofermenting
lactateand some acetate in additionto ethanol
and CO.

A. Homofermentative pathway
The homofermentative pathway is illustratedin Fig.8.2. A close rela-
fermentationisapparent.Glucose isdegraded via
tionshipto the alcohol
how-
the Embden-Meyerhof-Parnaspathway to pyruvate.The latteris,
ever,not decarboxylatedto acetaldehydeas in the alcoholfermentation
but used directlyas H-acceptor.The ATP yieldin both fermentationsis
the same, 2ATP/glucose.

B. Heterofermentative
pathway
in Fig. 8.3.As
The heterofermentativepathway is illustrated the
in
is formed via
oxidativepentose phosphate cycle,ribulose-5-phosphate
which is
yieldsxylulose-5-phosphate,
6-phosphogluconate.Epimerization
cleaved into glyceraldehyde-3-phosphateand acetyl phosphate by an

heglucose
2ATP
2ADP

2 lactate
2 glyceraldehyde-3-
2NAD 2P

2NADH+ 2H
2 pyruvate
21.3-bisphosphoglycerate

4ATP AADP)
Figure8.2.
Formation of lactate
from glueoseby thehomofermentative
pathway. 1,
Enzymes of the Embden-Meyerhof-Parnas pathway; 2, lactate
dehydrogenase.
 Lactate Fermentation 217

ethanol
NAD'

ADH+ H
acetaldehyde

COA NAD
NADH+ H* glucose
acetyl-CoOA

CoA ATP)
CH-CO-OPO3 H2 ADP)
acetyl-
glucose-6-(P
NAD
H3PO4
NADH+ +H
H3C-C-TPP-E TPP-E
6--gluconate
NAD
NADH+ H
H2C-C-TPP-E Co
OH ribulose-5-(P
HO
H2C-CH-TPP-E
OH OH
CH2OH

HO-C-H
H-C-OH
O-C-H
H--OH CH-0-
xylulose-5-0
CHa-0-
glyceraldehyde-3-

P NAD NAD
NADH+ H l
NADH+ H 2ADP
NAD (2ATP

lactate

Figure8.3.Formation ofCO2, lactate,

and ethanolfrom glucoseby the heterofer
mentative pathway. 1, Hexokinase;2,
glucose-6-phosphatedehydrogenase; 3,
6-phosphogluconatedehydrogenase;4, ribulose-5-phosphate 3-epimerase; 5,phos-
phoketolase.The cleavage reactionyields
glyceraldehyde-3-phosphateand en-
zyme-bound a,B-dihydroxyethyl-thiamine pyrophosphate. This is converted
acetyl-TPP-E via the a-hydroxyvinyl
derivative;
phosphorylytic cleavage resultsin
acetyl phosphate formation.6, phosphotransacetylase;7,
acetaldehyde dehy-
drogenase;8, alcohol dehydrogenase; 9,enzymes as inhomofermentative pathway.
 218 8: Bacterial
Fermentations

enzyme not mentioned thus far,phosphoketolase.Itcontainsthiamine

pyrophosphate and the formationof acetyl
phosphate can be understoodif
enzyme-bound a-hydroxyvinyl-thiaminepyrophosphate is assumed to
occur as an intermediate.
Acetylphosphate isconvertedintoacetyl-CoAby phosphotransacety.
lase.Subsequent reduction by acetaldehydeand alcoholdehydrogenases
yields ethanol. The
glyceraldehyde-3-phosphateformed in the phos
phoketolase reactionisconverted to lactateas in the homofermentative
pathway.
Inthe course of thisfermentation2NADH are formedand
the ATP consumed;
yieldis1 per mol of glucose-i.e., half of that of the
mentative pathway. homofer

C. Bifidumpathway
Inglucose breakdown by Bifidobacteriumbifidum two phosphoketolases
are involved:one specificfor fructose-6-phosphate
and one specificfor
xylulose-5-phosphate.The mechanism of both reactionsissimilar;fruc-
tose-6-phosphatephosphoketolase splits intoacetyl
fructose-6-phosphate
phosphate and erythrose-4-phosphate.

CH,OH CH-CO-OPO,H2
-O
HO-C-H H-C=O
H-C-OH H-C-OH
H-C-OH H--OH
CH-0-PD CH-0-P
The bifidum pathway is illustrated in Fig.8.4. Itexhibitsa
very
interesting
sequence of reactions.
W ithout the participation
ofhydrogena
tionand dehydrogenation reactions2 mol of
glucose are convertedinto
3 mol of acetate and 2 mol of
The conversion
glyceraldehyde-3-phosphate.
of the latterto lactateinvolves then
glyceraldehyde-3-phosphate and
lactatedehydrogenases.The formation ofacetate from acetyl
phosphate is
coupled to the formationof ATP from ADP.

acetate kinase
CH-C-0-PO,H2+ ADP CH-COOH + ATP

This reaction,
which iscatalyzedby acetate
kinase,isof greatimpor
all
tance for anaerobesthatform acetate because iteffects
ATP synthesisby
substrate-level
phosphorylation.With 2.5 mol of ATP per mol of glucose
the ATP yieldof the bifidumpathway is
higherthan thatof the homo- and
heterofermentative pathway.
Lactate Fermentation
219

2glucoxe
2ATP)
ADP
fructose-6- fructose-6-P

erythose-4- ucetyl-D
KADP)

glyceraldehyde-3-Psedoheptulose-7-Pacetate

xylulose-5- ribose-5-
5
ribulose-5-
6
xylulose-5-
2P

2 2
glyceraldchyde-3- acetyl-)
2P, 2NAD 2ADP
2NADH+ 2H ATR
2NADH+ 2H 4ADP 2 acetate
2NAD AATP

2 lactate
Figure8.4.Formation ofacetateand lactate
from glucoseby the bifidumpathway.
1,Hexokinase and glucose-6-phosphateisomerase;2, fructose-6-phosphate
phos
4,
phoketolase;3,transaldolase; transketolase;5,ribose-5-phosphateisomerase;
6, ribulose-5-phosphate3-epimerase;7,xylulose-5-phosphatephosphoketolase:
8,acetatekinase;9, enzymes as inhomofermentativepathway.

D. of lactatedehydrogenases
Stereospecificity
Table8.3has shown that lactic
acidbacteriaform either or
D(-)-,
L(+)»,
DL-lacticacid.D(-)-Lacticacid-formersproduce thisenantiomer exclu-
sively whereas L(+)-lacticacid-formersalways produce some
enantiomer.Most organismsthat excrete DL-lactate
D(-)
containtwo lactate
dehydrogenases that differin theirstereospecificity.
Some lactobacilli,
however, produce firstL(+)-lacticacid, which-whileaccumulating
inducesa racemase. This enzyme then convertsthe intothe
L(+)-form
. Butyrateand Butanol-Acetone
Fermentation
The fermentationof sugars to butyricacid was discoveredby Pasteurin
1861.Soon after,microorganismsresponsible forthe formationofbutyrate
were isolated,and itwas found thatseveralclostridial out
speciescarried
thistype of fermentation.Generally,only obligateanaerobes formbuty
rate as a main fermentationproduct;they belong to the four genera
Clostridium, Butyrivibrio,
Eubacterium, and Fusobacterium(Table 8.4)
 Fermentation
Butyrateand Butanol-Acetone 225

Table 8.4.Some species

forming butyrate as a
major fermentation end
product

Clostridium
butyricum
C. kluyveri
C. pasteurianum
Butyrivibrio
fibrisolvens
Eubacterium limosum
Fusobacteriumnucleatum

The mechanism of butyrateformation was not well understood until

Barkerand collaborators did theirpioneeringstudieson C. kluyveriand
untilferredoxin was discoveredin C. pasteurianum.The clostridia em-
ploy phosphotransferasesystems for sugar uptake and the Embden-
Meyerhof-Parnaspathway fordegradationof hexose phosphates topyru
vate.The conversion of pyruvatetoacetyl-CoA involvesan enzyme system
not discussedthus far:pyruvate-ferredoxin
oxidoreductase

A. Ferredoxin
and pyruvate-ferredoxin
oxidoreductase
When a cell extractof C. pasteurianumisincubatedwith pyruvate under
anaerobic conditionsthe decomposition of pyruvate according to
th
followingequation can be observed:

CH-CO-COOH + H,PO CH-CO-OPO,H2 + CO2 + H2

Acetylphosphate isformed and hydrogen gas isevolved.Thisreaction
isknown as the phosphoroclastic A thorough investigation
reaction. of the
enzymes involvedresultedin the findingthat this
phosphorylyticcleavage
isthe sum reactions;
ofseveral they are summarized inFig.8.7.Pyruvateis
first
decarboxylatedby pyruvate-ferredoxin oxidoreductase;the remaining
C2-moiety is covalentlybound to the TPP-containing as in the
enzyme
pyruvatedecarboxylaseand the pyruvate
dehydrogenase reactions.Inthe
next step acetyl-CoAis
formed;in contrastto pyruvate
the two hydrogens are not transferred dehydrogenase
to NAD, but are used to reduce
ferredoxin. Thisdifference
isimportantbecause ferredoxin
has a verylow
redox potential;at pH 7.0itisabout the same
as that of the
hydrogen
electrode

Ha/2H
red. ferredoxin/ox.
E = -0.42V
ferredoxin E =-0.41V
NADH + Ht/NAD
E6 =-0.32V
226
8: Bacterial
Fermentations

pyruvate+TPP-E HETPP-E+CO

2 HETPP-E + ferredoxin+ CoA TPP-E+ acety-CoA+ feredoxin H2

hydrogenase
H2
ferredoxin ferredoxin H2+|
phosphotransacetylase

acetyl-CoA+P acetylphosphate +CoA

Figure8.7.Steps ofphosphoroclastic
reaction.Steps 1 and 2 involvethe enzyme
oxidoreductaseand ferredoxin.
pyruvate-ferredoxin TPP-E, thiamine pyrophos-
oxidoreductase;
phate-containing HETPP-E, hydroxyethyl-TPP-E. Steps 3 and 4
are catalyzedby hydrogenase and phosphotransacetylase,
respectively.

Consequently, even in an environment saturatedwith hydrogen gas,

reduced ferredoxin can transferelectronsto hydrogenase and hydrogen
can be evolved. Thisiswhat isobserved when clostridia ferment carbohy-
drates.Moreover,the pyruvate-ferredoxin oxidoreductaseisreversible
and under appropriate conditionspyruvate can be synthesizedfrom
acetyl-CoA, reduced ferredoxin, and CO2 (see C. kluyveri).
NADH isa much weaker reducingagent thanreduced ferredoxin. This
isone reason why the pyruvatedehydrogenase reactionisnot reversible.

Ferredoxinwas discoveredin C. pasteurianum by Mortenson,Valen-

tine,and Carnahan in 1962. Itbelongs to the iron-sulfurproteins(until
recentlycallednonheme-iron proteins) and itshould be remembered that

thisclassofproteins playsan importantroleinthe respiratory chain(see

2.7and ferredoxins
Clostridial h ave a
Figs. 2.11). molecularweight of6000
and containeightironatoms permolecule, which are arranged inthe form
[4Fe-4S]-clusters. One of these
of two cubanelikeiron-sulfur clusters,
clustersisshown inFig.8.8.The ironatoms arebound to cysteineresidues

of the peptidechain and they are interconnectedby sulfidebridges.So

ferredoxincontainseightsulfide bridges;upon acidification of ferredoxin
solutionsthey are liberatedas HS. Thissulfuristhereforecalledlabile
sulfur.Clostridialferredoxinsare 2-electroncarriers(one electron

eys

cys

Figure8.8.[4Fe-4S]-cluster
of ferredoxin.,
Fe atoms; O, sulfur
atoms.
 Butyrateand Butanol-Acetone
Fermentation
227

Because ottheirbrOwnish colorthey are easily

cluster). recognizedincell
extractsand during fractionation of such extracts.
Itshouldbe emphasized thatnot allferredoxins have the same general
composition. The span of variationisapparent from Table8.5.Itcan be
seen that the C. thermoaceticum ferredoxin differsfrom the typical
ones in that it containsonly one
clostridial The Chroma
cluster.
[4Fe-4S]
tium ferredoxinis largerand the one of D. harbors
gigas even three
clusters.In E. coli,in A. vinelandi, and in many otheraerobes we find
iron-sulfurproteinswith 2Fe-2S clusters. The occurrenceof even 3Fe-
3S]clustersinsulfate-reducing bacteriahas been reported.
Allthe [4Fe-4S)-ferredoxins mentioned have in common that they are
low-potential electron carriers.The redox state of theirreduced form is
3Fe+ 1Fe and that of theiroxidized form is correspondingly
2Fe2+ 2Fe. Another classofiron-sulfur proteinispresent in many
bacteria,the high-potential ironproteinscalled HiPiP.Their redox poten-
tialis in the order of +350 mV; the HiPiP'sreduced form
contains
2Fe +2Fe and the oxidized form 1Fe + 3Fe. This difference
makes the ditference in redox
potentialunderstandable.
The investigation of compositionand structureof purifiedenzymes in
recent years has shown that many of them containiron-sulfur centersas
well.Examples are hydrogenase, formate sulfite
dehydrogenase, reduc
tase, pyruvate: ferredoxinoxidoreductase, CO2 reductase,nitrogenase,
CO dehydrogenase, CO oxidase,trimethylamine
dehydrogenase, dihy-
droorotate dehydrogenase, glutamate synthase, and xanthine
dehy-
drogenase.
One otherproteinisknown that isalso a electroncarrier
low-potential
but differsin other aspects:flavodoxin. Itisformed by severalobligate
anaerobicbacteriawhen they grow in iron-limited media. Ironand labile
sulfide
are absent and flavinmononucleotidefunctionsas redox group.The

Table8.5.Propertiesof variousferredoxins

molecular No. of
Organism weight cluster clusters E% (mV)
Clostridiumpasteurianum 6000 4Fe-4S 2 390
C. acidiurici 6000 4Fe-4S -430
C. thermoaceticum 7500 4Fe-4S ND
Chromatium vinosum 10,000 4Fe-4S 480
Desulfovibriogigas 18,000 4Fe-4S -455
Escherichiacoli 12,000 2Fe-2S -380
Azotobacter vinelandii 21,006 2Fe-2S -225
Chromatium vinosum HiPiPa 10,000 4Fe-4S 1 +35

HiPiP, iron
High-potential protein;ND, not determined
 228 8: Bacterial
Fermentations

redox potential liesaround -0.4V. Finally itshould be mentioned

that
rubredoxinoccurs in many anaerobes. This redox carriercontains
per
redox center one iron,which is bound to four
cysteineresiduesof the
polypeptide chain.Thus, rubredoxindoes not contain labilesulfide. Its
redox potentialliesaround -0.06V. The function of
rubredoxin in
anaerobes is unknown; in some aerobes itisinvolvedin
alkane oxidation
(see Fig.6.11).

B. The path of butyrateformation

The reactions
involvedinbutyrateformationfrom
glucoseare summarized
in Fig.8.9.The
Embden-Meyerhof-Parnaspathway and pyruvate-
ferredoxinoxidoreductase yield2
acetyl-CoA,2C02, and 2 reduced
ferredoxins,
which are reconvertedto the oxidizedform by
under hydrogenevolution. hydrogenase
Furthermore, 2NAD are reduced and 2ATP
are formed. The
advantage of hydrogen evolution inthe courseof
breakdown isapparent;only 2NADH are formed in the pyruvate
degradationof
glucose to acetyl-CoA.
The NADH formed in the
glyceraldehyde-3-phosphate dehydrogenase
reaction isoxidized by the conversionof2 acetyl-CoAintobutyrate.Inthis
pathway acetoacetyl-CoA, and crotonyl-
COA are intermediates. L(+)-B-hydroxybutyryl-CoA,
Itshould be remembered thatthe
storage material
poly-B-hydroxybutyric acidisa polymer of the
D(--form (see Fig.5.20)
and that the thioester of the
D(-)enantiomeris an intermediatein
long-chainfattyacid synthesis.
Butyrateis not formed from butyryl-CoAby simple this
would be a waste of energy.Instead hydrolysis;
butyryl-CoAisconvertedto butyryl
phosphate, and finally the phosphate group istransferred to ADP. These
reactionsproceedunder catalysis of the enzymes
and butyrate kinase, phosphotransbutyrylase
which are analogous to
phosphotransacetylase and
acetate kinase.
Figure8.9 shows that the ATP yieldof the
butyrate fermentationis
3ATP/glucose; thisismore than was gained inthe fermentationsdiscussed
thus far.

C. The formationof acetone and butanol

A number of butyrate producing clostridia
form small amounts of n-
butanol.With a few species,however, a realshiftfrom
to solvent production (n-butanoland acetone or butyrateproduction
isopropanol)can be
observedunder certainconditions.These
speciesincludeC. acetobutyli-
cum, C. beijerinckii,C.
tetanomorphum, and C. aurantibutyricum. The
most prominent speciesamong these isC.
acetobutylicum,which has been
used on an industrial
scale for the synthesis
of n-butanoland acetone from
molasses. Since n-butanol is very toxic (itinterferes with membrane
Butyrateand Butanol-Acetone
Fermentation
229

CH CH CH COOH
butyrate

ATP
ADP

CH,-CHCH-CO-0-(P)
butyryl phosphate ducone

2NAD 2P
CoA
2NADH+ 2H
ADE
CH-CH-CH, CO CoA
ATP
butyryl-CoA
NAD 24, 2Fd2CH,-CO-COOH
2CO,
NADH+ H' 2Fd 2CoA
CH-CH-CH-CO-CoA H2
crotonyl-CoA
2CH,-C0-CoA
H20
CoA
CH,-CH-CH-CO-CoA CH-CO-CH,-co-CoA
OH acetoacetyl-CoA
L(+)-B-hydroxybutyryl-CoA

NAD NADH+H
sum: glucose + 3ADP +3P butyrate+ 2c0,+2H2 t3ATP
Path ofbutyrateformationfrom glucose.1,Phosphotransferase
Figure8.9. system
and Embden-Meyerhof-Parnaspathway; 2,pyruvate-ferredoxin oxidoreductase
5,
(thiolase);L(+)-B-hydroxy-
3, hydrogenase; 4, acetyl-CoA-acetyltransferase
6, L-3-hydroxyacyl-CoA 7,
hydrolyase(crotonase);
butyryl-CoAdehydrogenase;
9, kinase.
butyryl-CoAdehydrogenase; 8, phosphotransbutyrylase; butyrate

low,in the
reached isrelatively
functions)the finalsolventconcentration
orderof 2%.
the courseof productformationduringgrowth of
Figure8.10 illustrates
C. acetobutylicum.Towards the middle of the fermentationwhen the pH
had dropped below 5, acidsare no longer produced, and n-butanoland
acetone appear as new products.During the solventphase the butyrate
concentrationdecreases again and an increaseof the pH is recorded.
constant.The shiftfrom acidto solventproduction
Acetate stays relatively
isnot only a response of the cellsto the low pH. Culturesgrowing very
Recent results
rapidlyoften"miss"the shiftand produce acids only.
are connected to one
of sporulation
indicatethat theshiftand the initiation
another.
 8:Bacterial
230 Fermentations

150

100

5o

10 20 30 40
Time (h)

Figure8.10.Course offermentationina batch cultureof C. acetobutylicumat low

phosphate concentration(0.62mM). Butanol,; acetone, A; ethanol,
rate,O; acetate,A; pH, O. [H.Bahl,W. Andersch,G. Gottschalk.
;buty-
Eur.J. Appl.
Microbiol. Biotechnol.15, 201205 (1982).]

The reactionsinvolvedin acetone and butanol formationare

summa-
rizedinFig.8.11. Under shiftconditionstwo enzyme activities
appear in
the cellsthat convert
acetoacetyl-CoA into acetone: a coenzyme A
transferaseand acetoacetate decarboxylase.Likewise,two
enzymes are
formed that reduce butyryl-CoAto n-butanol.The ratioinwhich
butanol
and acetone are formed liesin the orderof 2:1.

Table8.6.Fermentation balancesof clostridia"

C. butyricum C. perfringens C. acetobutylicum

products (amounts formed in mol/100 mol glucose fermented)

butyrate 76 34
acetate 42 60 14
lactate 33
CO2 188 176 221
H2 235 214
ethanol 135
26
butanol
56
acetone
22
aW. A. Wood, In: The Bacteria,I.
C. Gunsalus and R. Y.Stanier
Academic Press,New York and
(eds.).
London, 1961,vol.2, pp.
59-149.
Butyrateand Butanol-Acetone
Fermentation
231

2 glucose
4ADP+ 4P

4NAD 4ATP
4NADH+ 4H 4H, +4C0,
CH-CO-CH2-CO-CoA CH-CO-CH2-CO-CoA
acetoacety-CoA acetoacetyl-CoA

2NADH+ 2H acetate

2NAD
H;0 acetyl-CoA
CHy-CH-CH,-CO-CoA CH3-CO-CH2-COOH
butyryl-COA
acetoacetate

NADH+H
CoA CO
NAD'
CH-CH2-CH2-CHOo
butyraldehyde CH3-CO-CH,
acetone
NADH+ H-
NAD

CH-CH2 -CH-CH2 OH
butanol
sum: 2 glucose+ 4ADP+ 4P,
butanol+ acetone+ 4H,+5CO2 +4ATP
formation in step 2 not considered)
(acetyl-CoA
Figure8.11.Formation of acetoneand butanolby C.acetobutylicum.
1,Reactions
as inFigure8.9;2, acetoacetyl-CoA:
acetatecoenzyme A transferase;
3,acetoace-
tatedecarboxylase;4,L(+)-B-hydroxybutyryl-CoA
dehydrogenase,crotonase,and
butyryl-CoAdehydrogenase;5, butyraldehydedehydrogenase;6,butanol
dehyd-
rogenase.

The acetone-butanol
fermentationisan interesting
process,and itmay
become of economic importance again in the future.

D. Fermentationbalancess
Table 8.6 givesfermentationbalancesforC.
butyricum, C. perfringens,
and C. acetobutylicum.It can be seen
that the fermentationschemes
depictedinFigs.8.9 and 8.11are not
exactlyfollowed.C.
butyricum forms
acetateand some extra
hydrogen;C. perfringens
formslactate
and ethano
as do many
saccharolytic Inthe C.
clostridia.
the amount of H2 evolvedis acetobutylicumfermentation
largelydiminishedbecause ofthe reutilization
of butyratefor butanol
synthesis.
Incomplex fermentations such as the one carriedout
cum itis difficult by C. acetobutyli-
to judge whether the
hydrogen balance iseven or not.
 Butanediotkermentation
Mixed Acidand

IV. MixedAcidand Butanediol

Fermentation
iscarried out by the enterobacteria.
Thistype offermentation Organisms
belonging to the genera Escherichia, Salmonella, and Shigellaferment
succinic,and formic acids.Inaddition
CO2, H2, and
sugarstolactic,acetic,
Serratia,and
ethanol are formed. Speciesof the genera Enterobacter,
Erwiniaproduce lessacids but more gas (CO,),ethanol,and above all
large amounts of 2,3-butanediol. Two typicalfermentationbalances are
are
given inTable 8.8,and the pathways leading to allthese products
summarized in Fig.8.15.
Enterobacteriaemploy the Embden-Meyerhof-Parnaspathway for
hexose breakdown. The pathway leadingto succinatebranches offat phos-
are derivedfrom pyruvate.
phoenolpyruvate;allother end products
Threeenzyme systems act on pyruvate,and the amounts in which the fer-
mentationproductsare formed depend very much on the activityof these
enzyme systems.Inthe mixed acid fermentation large amounts of lactate
are formed by the actionof lactatedehydrogenase.Littlelactateonly
The two otherenzyme sys-
is produced inthe butanediol fermentation.
tems-pyruvate-formate lyase and a-acetolactatesynthase-deserve spe
cialattention.

A. Pyruvate-formate
lyase
are able to synthesize two enzyme
The enterobacteria systems foor

pyruvate breakdown to acetyl-CoA. The pyruvatedehydrogenase multien-

zyme complex is involvedin aerobic metabolism.Under anaerobic con-
ditionsitisno longersynthesized,and the enzyme still
presentisinhibited

Table8.8.Productsformed in the mixed acid and butanediol

fermentation"

Escherichiacoli Enterobacteraerogenes
product (mol formed/100 mol glucose fermented)

formate 2.4 17.0

acetate 36.5 0.5
lactate 79.5 2.9
succinate 10.7
ethanol 49.8 69.5
2,3-butanediol 0.3 66.4
CO 88.0 172.0
75.0 35.4
hydrogen

W.A. Wood, I.
In: The Bacteria, C. Gunsalus and R. Y.Stanier
Academic Press,New York and London,1961,vol.2,pPP. 59
(eds.).
149
 ghucose

(H)
ADP
suaccinate -oxaloacetate
ATP
PEP
- ADP

lactate Pyruvate
CoA

acetaldehyde 2 acetyl-CoA
formate

CoA
ethanol
acetyl-
ADP
CO
ATP

acetate

glucose

(H)
ADP
(H)
ATP
lactate Pyruvate

(H) 33
acetal
dehyde acetyl-CoA
formate
6(H)
ethanol

1 CO H
CO
a-acetolactate

co
ace toin

H)
2.3-butanediol
Figure8.15.Mixed acid(a)and butanediol
(b)fermentation.1,Enzymes of the
Embden-Meyerhof-Parnas pathway; 2, lactate
mate lyase;4, 3,
dehydrogenase; pyruvate-for
formate-hydrogen lyase;5,
acetaldehydedehydrogenase; 6, alcohol
dehydrogenase; 7,
phosphotransacetylase;8,acetatekinase;9, PEP
10,malate dehydrogenase, fumarase, and carboxylase
fumarate reductase;11, a-acetolactate
synthase;12,a-acetolactatedecarboxylase;13,2,3-butanediol
dehydrogenase
Mixed Acid and Butanediol
Fermentation
239

by NADH. Instead,the synthesisof pyruvate-formatelyase is induced

under anaerobicconditions. The reactioncatalyzedby this
enzyme pro-
ceeds intwo steps with an acetyl-enzymeas intermediate
and formateand
acetyl-CoA as products:

CH-CO-COOH + enzyme CH3-CO-enzyme + HCOOH

CH3-CO-enzyme + CoASH enzyme + CH-CO-SCoA

and
Pyruvate-formatelyaseisirreversibly rapidlyinactivated under air
that it infermentativemetabolismofthe enterobacteria.
so functionsonly
Apparently even under anaerobicconditionsthe activeenzyme isnot very
stable;at low concentrationsof pyruvate itchanges overto an inactive
form which can again be reactivated. This reactivationrequiresthe
reduced flavodoxin,activating
presence of four components; enzyme
(enzyme II),S-adenosylmethionine,and pyruvate.The latteris not
consumed in the activation thus,itfunctionsas positive
reaction; effector.

pyruvate-formatelyase pyruvate-formatelyase
(inactive) (active)

reduced flavodoxin enzyme II flavodoxin

(pyruvate)

S-adenosylmethionine methionine + 5'-deoxyadenosine

cleaved in thisactivation
S-adenosylmethionineis reductively reaction.
How and where the lyase actuallyismodifiedis not known.
The advantage of the pyruvate-formateIyase over the pyruvatedehy-
drogenasecomplex infermentative
m etabolismis apparent:the formation
of acetyl-CoAis not accompanied by the reductionof NAD*.

B. a-Acetolactate
synthase
The thirdenzyme system actingupon pyruvate isa-acetolactate
synthase.
isalso
Thisenzyme formationby bacilli
involvedin 2,3-butanediol (see
Fig.6.26). It containsthiamine pyrophosphate. First,enzyme-bound
hydroxyethyl-thiamine pyrophosphate and CO2 are formed from pyru
vate. Active acetaldehyde is then transferred
to a second molecule of
Pyruvate:

CH-CO-COOH + (H)TPP-E CH-CH-TPP-E+CO

OH
CH-CH-TPP-E
OH CH-CO
+
+(H)TPP-E
CH-CO-COOH CH-C-CoOH
OH
This synthase isformed and isactiveunder slightly
acidicconditions,
and itisreferredto as the 6
pH enzyme. Thus, a decrease of pH in the
240 8: Bacterial
Fermentations

environment of Enterobacteraerogenes leads to an increase of 23.

butanediolformation.Consequently less acids can be produced from
Pyruvate.
The pH 6 enzyme isdistinctfrom the anabolic a-acetolactatesynthase,
which isinvolvedin valinesynthesis.This enzyme ismost activeat pH 8
(pH 8 enzyme) and is subject to feedback inhibitionof-valine

C Formate-hydrogenlyase
Species belonging to the genera Shigellaand Erwiniado not contain
formate-hydrogen lyase;they produce considerableamounts of format
Escherichiacoli contain this activitywhen
and Enterobacteraerogenes
grown on sugars under anaerobic conditions,and formate can be cleaved
intoCO and H2.
is The formation
Formate-hydrogenlyase not one singleenzyme entity.
of H2 and CO% from formate is the resultof the combined of a
activity
special formate dehydrogenase (FDH1) and a hydrogenase. FDH, is
under is observed only, if this
redox control,and formate cleavage
compound cannot functionas electrondonor for the nitratereductase or
fumarate reductase.Inother words, ifnitrateor fumarate is
present,
formate isoxidizedby another formate dehydrogenase (FDH,), and from
there the electronsare channelledto nitrateor fumarate, but not to
H
(Fig.8.16). Thus,H2 evolutionisnot observedinthe presenceof nitrate or
fumarate. o

Allthe enzyme systems mentioned above are membrane-bound. The

formate dehydrogenases are selenoproteinscontainingiron-sulfurcenters
molybdenum in the form of Moco-factorand cytochrome b.

D. coupledto
Decarboxylations ebslotsaA
membrane energization
Likeseverallactic
acidbacteria,
some enterobacteria
areableto grow with
citrateunder anaerobicconditions. Citrateiscleavedby citrate lyase,and
the oxaloacetateformed is to
decarboxylated yieldpyruvate (see section
II.G of thischapter).Sternand collaborators showed a number ot years
ago that growth of Enterobacteraerogenes on citratedepended on the
resenceof sodium ions,Dimroth demonstrated that Na" was
requiredby
the oxaloacetate
decarboxylasewhich is a biotin-containing and mem-
brane-associated enzyme. Interestingly enough thisenzyme manages to
couplethe decarboxylationreactionwith the generationofan
electrochem
icalgradientof sodium ions as depictedin
Fig.8.17. This gradientcan be
transformedinto a pH gradientthat can be takenadvantage of
by the ATP
synthase.The decarboxylationof oxaloacetateis associated with a free
energy change of AG=-30 kJ(-7.2 kcal)mol, and one could
expectsynthesisof 1/3 ATP per 1 CO, formed. The uptake of 3 H per
 Mixed Acid and ButanediolFermentation
241

NO
nitrate
reductas

eytbss

UO
HCOOH

FDH Fe-S eytb 2e

2H+CO MK

cytb
fumarate

fumarate
reductase

succinate

HCOOH
H
FDH Fe-S cytb
2e hydrogenase

2H'+CO
Figure8.16.Formate-hydrogen lyase reactionand relationshipt9itrate and
fumarate reductases.Only when NO5 or fumarate isnot
available H, formed.

oxaloacetate

Na
CO, tpyruvate

Na

H
nADP + nP

HATP

Figure8.17.
Sodium-dependent oxaloacetatedecarboxylase.a: Sodium transloca-
tion as
coupled to the decarboxylation reaction.b: Na-H
antiporter
c: ATP synthase;nmay be in the order of 1/3.
Proton-translocating
242
8: Bacterial
Fermentations

ATP synthesizedwould be in agreement with this Three remarks

figure.
are necessaryin thiscontext:

1.Anaerobic citratedegradation in most organisms is not sodium-

dependent. This is true for lacticacid bacteria,
clostridia. phototrophs,and
Here, oxaloacetatedecarboxylase is a soluble
enzyme not
containingbiotin. E. coliis able to utilizecitrate
anaerobicallyin the
presence of a cosubstratesuch as
glucose; itdoes not
contain
tatedecarboxylase,and the Oxaloacetateformed isreduced oxaloace
tosuccinate
(withthe reducingpower from the of
degradation glucoseto acetate).
Thus,the sodium-dependent oxaloacetate
to be decarboxylasedoes not seem
widespread among the anaerobes.
2. Other
decarboxylationreactionsare coupled to Na translocation as
well.This has been shown for
methylmalonyl-CoAdecarboxylaseof
Veillonellaalcalescens
and glutaconyl-CoA
decarboxylaseofAcidami-
nococcus fermentans and Clostridium
symbosium.

methylmalonyl-CoA propionyl-CoA+CO
Na
glutaconyl-CoAcrotonyl-CoA CO +
The enzyme mentioned first in propionate fermentation,
participates
the second one in a
pathway for the anaerobic breakdown of L
glutamate.
of
Decarboxylation methylmalonyl-CoAisthe onlyenergy-yielding
reaction,
when Propiogenium modestum on
grows succinate:
Succinate +Ht propionate+ CO2 AG= -20.6kJ/mol
3. We have seen that not
only substrate levelphosphorylationisused by
anaerobes forATP synthesisand not
only ATP hydrolysis forenergiza-
tionof the membrane. Some lacticacid
bacteriatake advantage of
product effux forthe generationof an electrochemical
anaerobes employ gradient;here,
decarboxylationreactionsfor thispurpose.
The next sectionsof this
chapterwill
show that electrontransportisalso
used by several of
groups obligateanaerobes forthe generationof a
protonmotive forceacross the membrane.

V. and SuccinateFermentation
Propionate
Propionateisa majorend
productof fermentationscarriedout by a variety
of anaerobic bacteria.
Many of them ferment glucose to
acetate,and propionate,
CO

A preferred
1.5glucose 2 propionate + acetate + COD
substrateof propionate-formingbacteriais so
lactate, that
 and Succinate Fermentation
Propionate 243

these organisms can grow with the major end

product of the lactate
fermentation:
3lactate 2 propionate+acetate+ CO
There are two pathways forpropionate formationfrom lactate; in the
acrylatepathway lactateis reduced stepwiseto propionate;in the succi
nate-propionatepathway lactateisconverted to propionateviapyruvate
and succinate.

A. The acrylate
pathway
This pathway seems to occur only in a few
microorganisms,e.g.,in
Clostridiumpropionicumand in Megasphaera elsde-
(Peptostreptococcus)
nii.
Itisshown in Fig.8.18,L-,D, or DL-lactate
may serve as substrate;a
racemase is present which interconvertsthe enantiomers. L-Lactateis
onvertedto L-lactyl-CoA ina CoA transferasereaction.By reactions
not
yet establishedin detailacrylyl-CoAisformed. Itisreduced to
and propionate is
propionyl
CoA, produced by the above-mentioned CoA trans-
ferase

OH OH

(L)2CH3-C-coOH 2CH-C-Co-CoA

2H,0
(D)CH- COOH
2CH, =CH-co-CoA
OH

ETF

ETF H2
CH-CO-COOH
CoA Fd ETF H2
FdH, ETF
CH3-CO-CoA 2CH,-CH2-CO-CoA

ADP
ATR)
CH-COOH 2 CH-CH3-COOH
sum:3lactate 2propionate+ acetate + CO +H,O
Figure8.18.Formation of propionate,acetate,and
CO2 from DL-lactateby
Megasphaera elsdeniiand Clostridium
propionicum. 1 ,Lactate racemase;2,CoA
transferase;
3, reactionnot established;
4, dehydrogenase,which employs reduced
electron-transferring
flavoprotein(ETFH2) as H-donor; 5, D-lactatedehy-
drogenase;6, pyruvate-ferredoxinoxidoreductase;7, transhydrogenase;8, phos-
photransacetylase+ acetate
kinase
 8: Bacterial
Fermentations
244

reduction isreduced electron-transferring

The H-donor foracrylyl-CoA
Itisformed from D-lactateand from reduced ferredoxin
flavoprotein.
(or
this fermentation is 1 mol/3 mol of
The ATP yield of
flavodoxin).
lactate.
C. propionicum also ferments alanine and acrylateto propionate.

B. The succinate-propionate
pathway
This pathway is employed by most propionate-producingorganisms.
Succinateisan intermediatebut isalsoproduced as end product insmallor
the acrylatepathway
largeamounts. On the otherhand, organisms using
do not excretesignificantamounts of succinate.
The establishmentof the succinate-propionate pathway was a rather
task.As isshown inFig.8.19severalenzymes are involved.First,
difficult
as
lactateisoxidizedto pyruvate in a reactionrequiringa flavoprotein

CH-CH2-COOH CH-CHOH-COOH

CH-CH2-C0-CoA CH3-CO-COOH
biotin-CO2

CHs biotin

HOOC-C-CO-CoA(S)

H
HOOC-C-c0-CoA(R)
HOOC-CH2-CO-COOH
CHs
B12-enzyme NADH+ H
NAD
HOOC-CH2-CH-CO-CoA HOOC-CH2-CHOH-cOOH

2H
HO
HOOC-CH-CH2-COOH HOOC-CH-CH-COOH

ATP+ H,OADP + P
sum: lactate+ NADH+ H'+ADP+P propionate+NAD + ATP+ 2H,0
Flgure 8.19.
Fermentationof lactatevia the succinate-propionate
pathway by
1,Lactate dehydrogenase (the H-acceptor is probably a
propionibacteria.
favoprotein);2, (S)-methylmalonyl-CoA-pyruvate 3, malate
transcarboxylasé;
dehydrogenase; 4, fumarase; 5, fumarate reductase;6, CoA transferase:7,
(R)-methyvlmalonyl-CoAmutase; 8, methylmalonyl-Co-A racemase.
 and SuccinateFermentation
Propionate 245

H-acceptor.Oxaloacetate isthen formed ina reaction

transcarboxylation
with(S)-methylmalonyl-CoA as CO-donorand biotinas CO-carrier.The
actionof malatedehydrogenase and fumarase
yieldsfumarate,which is
reduced to succinateby fumaratereductase. This reductionreactionis
coupled to ATP formationby electrontransport Suc-
phosphorylation.
is
cinyl-CoA then formed ina CoA transferase reactionand the
ment as catalyzedby the rearrange
coenzyme B12-Containing methylmalonyl-CoA
mutase leadsto
(R)-methylmalonyl-CoA,which is not a substrateforthe
transcarboxylase.Rather,the (S)-enantiomer is formed by a specific
racemase.Then transcarboxylation
yieldspropionyl-CoAand CoA trans-
fer to succinatefinally
yields
propionate
One NADH isconsumed in formationfrom lactate;
propionate itcomes
from lactateoxidation
to acetate accordingto the overall
fermentation
equationgivenabove.
Besidesthe
transcarboxylase-a enzyme with a high
biotin-containing
molecularweight
(approximately
800,000 daltons)and a very complex
quarternarystructure-two enzymes of the succinate-propionate
path-
way deserve special
attention:
methylmalonyl-CoAmutase and fumarate
reductase.
VI. AcetateFermentation
We have seen that acetate isan inportantproduct in a number of
fermentations.There isa group oforganisms,however,by which acetateis
formed as the predominantnongaseous product.Butyrateisusuallynot
detectablein the fermentationbroth;ethanoland lactateinvery small
amounts at the most. This formerlyvery small group of acetogenic
organismshas grown verymuch inrecentyears.Representative speciesare
given inTable8.9. Itisobviousthat most speciesare able to liveat the
expense of acetateformationfrom H2 and CO2 accordingto the following
equation:
4H2 +2CO,- CH-COOH + 2H,0
AG =-107.1 kcal)per mol acetate
kJ (-25.6
in1936 by Wieringawho isolated
was discovered
Thistype of fermentation
aceticum.Severalacetogensgrow with carbon monoxide and
Clostridium
 250
8:Bacterial
Fermentations
Table 8.9.Acetogenic bacteriaand some of their
properties

growth on
organism
thermophilic H +
CO sugars cytochromes
present
Clostridiumaceticumn
C.
thermoautotrophicum
C.
formicoaceticumn
C. thermoaceticum
Acetobacteriumwoodii
A. wieringae
Acetogenium kivui
Sporomusa sphaeroides

Some strainsare
positive.
Eubacteriumlimosum isableto
amounts of produce acetate from + H2,but it
butyratewhen CO forms
growing on other substrates. considerable
with methanol
plusCO:
4CO+2H,0 CH-COOH +2C0
AGO -484.4kJ (-115.9
kcal)perreaction
4CH,OH+ 2C02
3CH-COOH
2H,0 +
AG=-706.3kJ(-168.9
kcal)
Hexoses are converted perreaction
by acetogenic bacteria
per mol: to almost 3 mol
acetate

The
CH206 3CH-COOH
recently described
genus Sporomusa
Gram-negative represents the first
utilize endospore-formers.Inadditionto genus of
N-methylcompounds H2 2CO, its +
members
n-butanol). (e.g.,betaine)and primaryalcohols
How isit (ethanol,
possiblef orthe
from one molecule acetogens to make three
of hexose? The moleculesofacetate
the hexose is pathway isdepictedin
Parnas
degraded to two Fig.8.23.First,
pathway.Their pyruvates via the
ferredoxin subsequent degradation Embden-Meyerhof
oxidoreductase and the by the actionof
2
yield acetates.In typical pyruvate;
enzymes actingon
addition, CO2 is acetyl-CoA
ferredoxin are produced,and NADH and
ofthe third generated from the reduced
moleculeof acetateiscorresponding oxidizedforms.
the way to now initiated Synthesis
by the reductionof CO all
5-methyltetrahydrofolate. The first
remarkableformate enzyme of this
It isa pathway isa
structured so as to dehydrogenase.
make formate
and nottungsten-selenoprotein and is
membrane-bound formate only to oxidizeitlikethe
terobacteria).The electron dehydrogenases of other
donor is known for organisms (e.g., en-
organisms;inC. thermoaceticum it only some
is NADPH. acetogenic
is
Formyltetrahydrofolate
 251
Fermentation
Acetate

fructose

2ADP+2P 2NAD
2ATP 2NADH+ 2H

pyruvate
Pyruvate

CoA- Fd COAN-Fd
FdH FdH

CO2 acetyl-CoOA CO
acetyl-CoA

ADP+P ADP+P XH
XH2

CoA
ATP CoA- ATP
10

acetate
acetate x+H,O

formate CO
ATP H,F CoA
methyl-B2-E
ADP+ P
11
10-formy1-H,F
acetyl-CoA

H,0
- ADP+

ATP
5, 10-methenyl-H,F COA

NADH+ H'- ace tate

NAD
5, 10-methylene-H,F
FdH
Fd-
S-methyl-H,F B2-E
via
Figure8.23.
Pathway of the acetate 1
fermentation. ,Degradationof fructose
oxidoreductase;
the
Embden-Meyerhof-Parnas pathway; 2, pyruvate-ferredoxin
formate dehydrogenase; 5,
5, plus acetate kinase;4,
phosphotransacetylase
synthetase; cyclohydrolase;
6, methenyl-tetrahydrofolate
formyl-tetrahydrofolate re-
dehydrogenase; 8, methylene-tetrahydrofolate
1,methylene-tetrahydrofolate
10, CO dehydrogenase;11,
ductase 9, tetrahydrofolate:
B12 methyltransferase;
enzyme (probablyATP-requiring);
acetyl-CoA-synthesizing [CO],enzyme-bound.

and subsequent
then formed from formate inan ATP-consuming reaction,
reductionleadsto the previouslymentioned methyltetrahydrofolate.
How acetate was the subject
issynthesizedfrom methyltetrahydrofolate
to intenseresearch in the laboratoriesof Wood and of Thauer. The
breakthrough came with the discovery of carbon monoxide dehydro-
genase-a nickel-containing enzyme. Itis differentfrom CO oxidase,
enzyme of the carboxydobacteria, that the enzyme-
the characteristic in
specificelectron carrierhas a low redox potential. That reaction 1s,
 Fermentations

reversible
therefore, under physiological
conditions:

CO2 +X-H CO +X + H,0

Inthe pathway, CO2 isnow reducedto CO, and itisthe latter which finally
gives the carboxylgroup of acetate:methyltetrahydrofolate iscarbony
lated,and acetyl-CoAand finally acetateare formed.
The pathway as depicted in Fig.8.23 also allows to outline
the routes
used foracetate formationfrom methanol + and from
CO, H2 + CO,.
The strategyisto make CO from CO and to make
methyltetrahydrofolate
from H2 + CO2 or methanol. Thus,
part of the methanol has to be
oxidizedto provide the reducing power for
CO,
reduction to CO0:
and CO finally
methyltetrahydrofolate yieldacetate.The energeticsof the
latterfermentations is not clear
yet. Likewise,the function of the
cytochromes in acetogens isunknown.

VII.Methane Fermentation
Methane isthe most reduced organic
compound and itsformationisthe
terminal step of the anaerobicfoodchain that willbe discussedin Section
IX of thischapter.Methanogenesis isa domain of the
Archaebacteria;in
otherwords,allknown
methanogenic bacteriabelong to thiskingdom of
organisms.Some of theircharacteristicfeatureshave alreadybeen outlined
in Chapter 5.

A. Substrateutilization
Methanogenic bacteriaare extremelyoxygen-sensitive. Thisisnot a great
disadvantage to them in nature.In habitats richin
degradable organic
compounds, oxygen is consumed rapidlyand trapped by
surfacelayers. organisms in
Thus the methanogenic bacteriaare
abundant
particularly
in allsorts of mud and sediments.
Other importanthabitatsof these
bacteriaare the rumen and the
(man-made) anaerobicdigestersinsewage
plants.The oxygen sensitivity of the methanogens creates
problemswhen
pure cultures are to be isolated
and experimentswith pure culturesare to
be carriedout.
Appropriatemethods (theso-called Hungate technique)
have been worked out for this
purpose.
Representatives of various
genera of methanogenic bacteria, the sub-
stratesutilizedby them, and the distribution of cytochromes are summa-
rizedinTable 8.10.Itis apparent that
complex organiccompounds cannot
be utilizedby the methanogens. Substratesare
C compounds and as the
only C2 compound: acetate.Two nutritional
groups of organisms can be
envisaged:
 253
Methane Fermentation

of the methanogenic bacteria,the substrates

Table8.10.Representativespecies
by them, and the presence of cytochromes
utilized

presence of
substratesutilized cytochromes
organism

Methanobacterium H2+ CO,CO°

thermoautotrophicum
Methanobrevibacterarboriphilus H2 + COD2

Methanococcus vanniellii H2 + CO2, HCOOH

Methanospirillumhungatei H2 + CO2, HCOOH
Methanosarcinabarkeri H2 + CO2,CH,OH
CH,COOH,
methylamines
Methanosarcinamazei CH,OH, CH,COOH,
methylamines
Methanothrixsoehngenii CHCOOH
Methanolobus tindarius eCH,OH, methylamines
Methanococcoidesmethylutens CH,OH, methylamines
Methanoplanus limicola H2 + CO2, HCOOH

CO allowsonlyslow growth;itmay also be used by other methanogens listed

1.Obligate chemolithotrophicmethanogens that grow with CO2 + H2

accordingto the equation:

CO2 + 4H CH+2H,0 AG =-136kJ(-32.4

kcal)
Some of these organisms alsogrow withthe ""quasi-chemolithotrophic"
substratesHCOOH and CO. Thisterm isadequate here because both
viaCO + H2:
substrates are utilized

4HCOOH 4CO2 + 4H2 (carrier-bound)

CO + 4H CH4 +2H,0
4HCOOH CH+ 3CO + 2H,0
AG=-144.2
kJ(-34.5
kcal)per mol methane

correspondingly:

4CO+2H,0 CH, +3CO,

AG=-211 kJ (-50.4kcal)per mol methane
2. Methylotrophicmethanogens that
grow with methyl-group-containing
substrates(methanol,methylamines,
acetate).The fermentation
equa
tionforacetate isvery simple:

CH-COOH CH,+ CO
AG=-37 kJ (-8.9
kcal)per mol methane
 Bacterial
254 rermentations

Organisms such as Methanosarcina barkeri grow on methano or

methylamines. Here,one-fourthof the substratehas to be oxidizedto
CO forreducingpower generation:

CH,OH + HO CO2 +6H

3CH,OH+6H- 3CH,+3H,0
4CH,OH 3CH + + 2H,0
CO2
AG =-319.5 kJ (-76.4
kcal)per reaction
AG (permol methane)-106.5 kJ (-25.5kcal)
or

4(CH,)s-N
+6H,0 9CH+ 3C0, +4NH
AGO -683.2 kJ (-163.4kcal)per reaction
AG (permol methane) =-75.9 kJ (-18.1 kcal)

Group organisms produce methane directlyfrom the methylgroups

2
and not viaCO2. Thishas been demonstrated with deuteratedsubstrates
thatgave methane withthreedeuteriums in it:

CD,OH mniao,CD,H
Obligatechemolithotrophicmethanogens do not containcytochromes,
which are,however,presentinmethylotrophicmethanogens. The metha
NaT ofabout 5 mM forgrowth;
nogenicbacteria require inconcentrations
itsfunction A number ofratherunique redox carriers and
isstill
unknown.
tooccur of
coenzymeshave been found inall these organisms;they do not
ocCur inother organisms(few exceptions)and are specifically
involvedin
the
pathway from CO to CH4. Itshouldbe mentioned that most of these
were Methanobacterium thermoautotrophicum,
first
isolated from
organism that was describedby Zeikus and Wolfe and that grows
mineral medium with H2 + CO2 at 55°C.

B. Novelcoenzymes
The firsttwo novelcoenzymes discoveredin methanogens by Wolfe and
co-workers were coenzyme M and coenzyme Fa20. Theirchemicalstruc-
turesare given inFig.8.24.Coenzyme M isa simplechemicalcompound.
Itsreactivegroup is the mercapto group which can be methylated and
methyl-coenzyme M isthe ultimateprecursorof methane. Coenzyme F420
inposition
isa deazaflavin(ring-N 5 of the flavin
skeletonismissing); itisa
redoxcarrier withan E6 =-370 mV, and itsroleisanalogous to the one
of ferredoxininotheranaerobes. Itfunctionsas electronacceptor of
hydrogenase and as electron donor in severalreductionreactions.
The
structureoffactorFa30 which
isinvolved inthefinalstep of methane
formation was elucidatedby Thauer,Eschenmoser,and co-workersas a
withnickelas centralmetal ion.Inaddition tohydrogenaseand
tetrapyrrol
CO dehydrogenase, both of which are nickelproteins, factorFa30 repre
 Methane Fermentation
255

CO0 CO0
CHCH-CH-ÇH-CHy
OH OH OH 0--o-CH--NH-CH-CH-CHy-NH-CH

HO CH
COo
coenzyme Fa20

ÇO0

H,NOC

HS-CH-CH-SO N= COo
H,C
coenzyme M Ni CI0
H
"OOC

o
CO0
factorF430

Çoo
oo
-NH-CH-CH,)-C-NH-CH-(CHa)-NH-CH,)» -oCH
CH,-NH

CH)
OCC-CH
CH-co0 methanofuran

H)-COOo

COO
HN
CH
CH2
HCOH
O= -0 -CH

HN CH
fH CH HcOH CH
H
HCOH H COO
H,N CH
H CH-o

5,6,7,8-1tetrah
ydrome thanop terin

Figure8.24.Novel coenzymes inmethanogenic bacteria.

sents thethirdcomponent of methanogens
containingthismetal. Two
additionalstructuresare given in Fig.8.24: that of
tetrahydromethano
pterindeterminedby Vogelsand co-workers, and of methanofuran. Both
are involvedin CO2 reduction.Methanofuran
functionsas the primary
CO acceptor.
 8: Bacterial
256 Fermentations

C. Pathway of methane formation

formationfrom CO, + H2. A
Firstwe shalllook at methane ence
scheme of methane tormation from CO2 is given in
based on Barker's
carriermolecule known to be involvedis methano-
Fig.8.25.The first
furan.In a reactionthat requires CO2 and reducing equivalentsit is
with the formylgroup residingat the
convertedto formylmethanofuran
furan ring.Transfer of the C moiety to
aminomethyl group of the
and reductionof the formylto the methyl group
tetrahydromethanopterin
follows. Inanalogy to the tetrahydrofolatebiochemistrythe methyl group
istransferred to coenzyme M
residesat N° of the ringskeleton.Itfinally
M reac-
and reduced to methane. The methyl-coenzyme methylreductase
tionhas been studiedindetail;the responsibleenzyme isof a verycomplex
itcontainsseveral factorFa3o and an as yetunidentified
structure; proteins,
factorB.

CO
MF-H

H F420

H,O
MF-CHO
THMP-H
Fe20
H2 MF-H
THMP-CH,OH

H F420

H,0
THMP-CH

-CoM-SH
THMP-H
CoM-S-CH

Fa20
IV H2

ATP

of ATP synthesis.
Figure8.25.Scheme forthe reductionof CO to CH, and site
1,
MF-H, Methanofuran;THMP-H, tetrahydromethanopterin; methyl-coenzyme
M methylreductase.
 Methane Fermentation
257

The freeenergy changes and the redox of the fourreactions

potentials
leadingfrom bicarbonateto methane are as follows:

HCO + H2 AG AEG
HCOO+ H,O -1.3 kJ (-0.3kcal)-432mV
HCOO+H2 + H CH,O+ H,O +23.0kJ( +5.5 kcal)-535mV
CHO+ H CH,OH -44.8kJ (-10.7 mV
kcal)-182
CH,OH+ H CH,+H,0 -112.5kJ (-26.9
kcal)+169 mV

HCO + H +4H2 CH+3H,O -135.6kJ (-32.4kcal)

(valuesper mol product)

Itis obvious that the firsttwo steps are not very favorable. The third
step isdistinctlyexergonic;most of the energy,however,isreleasedinthe
fourth step. The values given are for the free redox
couples (e.g.,
CH0/CH, OH). They might be slighlydifferent for the carrier-bound
compounds.
Itcan be stated now that the fourthreductionstep is
coupled to the
generationof a protonmotive forceat the membrane which inturnisused
by an ATP synthase forthe phosphorylation of ADP. The evidencecomes
from experiments with Methanosarcinabarkeri.Reduction of CH,OH to
CH by H2 is coupled in thisorganism to an increaseof the intracellular
ATP concentration.This ATP synthesis and alsomethane formationare
inhibitedby dicyclohexylcarbodiimide (DCCD)-the classicinhibitor of
the ATP synthase (Fig.8.26).The protonmotive force,AP, however,
remains unaffected(because itcannot be utilized forATP synthesisinthe
presence of DCCD). The two processes-methaneformationand ATP
synthesisare apparentlycoupled via AP.As expected,thiscouplingcan
be abolishedby an uncoupler. In itspresence,the protonmotiveforceis
dissipatedand methane isproduced againfrom CH,OH + H2, but ATP is
not synthesized. The sequence of effectsis therefore:methane
formation protonmotiveforce ATP synthesis and not: methane
formation ATP synthesis-protonmotive force.ItexcludesthatATP
isformed by substrate-level phosphorylation.Since methane isproduced
from methanol + H2 in one reduction step-in the methylreductase
reaction-thisstep must be the site of energy conservationby a che-
miosmotic mechanism.
Methanosarcina barkeriis the classic, and representativespeciesfor
those methanogenic bacteriathatutilize acetate,methanol, and methyla-
mines as substrates. Growth on acetate ismuch sloweras compared tothat
on the other substrates.Nevertheless, it isthe most important metha
nogenic substrateinnature (seeSectionIX).Lookingat the fermentation
equation may give the falseimpression thatmethanogenesis from acetate is
a After the discovery of high levelsof CO
simple decarboxylation.
 8: Bacterial
258
Fermentations

CH,OH+ H DCCD uncoupler

protonmotive force

LATP

CH
formation

time

Couplingof ATP synthesis

Figure8.26. to methane formationfrom CH,OH + H2
in M. barkeriby a chemiosmotic mechanism [Redrawn from M. Blaut and
G. Gottschalk;Eur.J.Biochem. 141,217-222(1984)].

dehydrogenase in acetate-growncellsof M. barkeriby Zeikus and co-

itbecame more and more clearthat CO isthe primary cleavage
workers,
not
productand CO2. The fermentationscheme inFig.8.27shows that the
can then be writtenas an oxidoreduction process:firstCO
fermentation
are produced,the oxidationof the former
and methyl-coenzyme M pro
videsthe reducingequivalents for the reductionof the latterto methane.
the it that the formationof
From
1
freeenergy change
from
(-31 kJ) isapparent
acetatecan yieldonly a fraction of a mol ATP.
mol methane
Ithas already been mentioned that
methanogenesis of methanol and of
the methylamines can be subdividedinto two
processes: oxidationof
one-fourthof the methylgroups to CO and reductionof three-fourths of
the methyl groups to CH (Fig. 8.28). A methanol: M and a
coenzyme
trimethylamine: coenzyme M methyltransferasehave been characterized.
The methyl group is firsttransferredto
protein-bound 5-hydroxy-
benzimidazolylcobamide which isthe principalform of Bi2 inmethanogens
(not the dimethyl derivativeas in the propionibacteria). Methyl group
transfer proceedsfurtherto coenzyme M. The carrier at which the methyl
group isoxidizedto CO, isunknown. Very likely the cytochromes
present
in those methanogens that grow on methanol and
methylamines are
involvedin the first oxidationstep (X-CH3 to X-CH,OH).
Coupling
of this oxidation(E=-182 mV) with reduction of factor
Fa20
(E%=-373 mV) requiresreverse electrontransferthat might procee
with the participation of cytochrome
 Methane Fermentation 259

CH-COOH

HX
H,O
CH-CO-X

ICO CoM-S-CH
H,0
2H CoM-SH

ATP
CO CH
8.27.
Figure Tentativescheme forthe formation ofmethane and carbon dioxide
from acetate.
1,methyl-coenzyme M methylreductase;2, CO dehydrogenase.
Detailsof the other reactionsare not yet known.

2H

H,0

X-CHO
2H

X-CH,OH XH
2H

H,O
X-CH3

H,0
3CoM-S-CH 4CH,OH

3H,0 3CoM-SH

ATP
3CH,
Figure8.28.Tentativescheme forthe formationof methane and carbon dioxide
from methanol. 1,Methanol: coenzyme M via Bi2;see
methyltransferase
(transfer
text);X, unknown carrier.
260 8: Bacterial
Fermentations

D. Synthesisof cellcarbon
M. thermoautotrophicum and a number ofotherspeciesare able to grow in
a mineralmedium. Therefore, these organisms must be able to make all
theircellularconstituentsfrom CO. Fuchs and co-workershave shown
thatthe methanogens do not operate a cyclicCO, fixation
pathway as do,
forexample,the phototrophs (seeChapter9).The strategy isto synthesize
acetyl-CoAfrom2CO,.For thispurpose
first
theorganisms use a pathway
that resembles the one present in acetogens (Fig.8.23).Advantage
is
taken of the factthat carrier-boundmethyl groups are intermediatesof
methane formation anyway. Not allof them are used to produce
methane; some are carbonylatedto give acetyl-CoA. The latterisreduc
tivelycarboxylatedtoyieldpyruvate. Ferredoxinor coenzyme Fa20 Serve as
H-donor. PEP synthetase and PEP carboxylase lead to oxaloacetate
that isreduced to succinate;in M. thermoautotrophium,finally succinyl-
CoA is reductivelycarboxylated,and a-oxoglutaratethe prcursor of
formed:
L-glutamate-is
succiny1-SCoA +CO2 + FdH2 a-Oxoglutarate+ CoASH + Fd
Thus,incontrastto Clostridiumkluyveri which alsouses acetyl-CoA as
startingmaterialforbiosyntheses,several methanogens lackcitrate
thase, and employ the "dicarboxylicacid portion" of the citricacid
cycleforglutamate synthesis ratherthan the "tricarboxylicacidportion"
M. barkeri, however, has been shown to containcitrate synthase and to
employ the "tricarboxylicacid portion" of the cycle.

VIll.SulfideFermentation(Desulfurication)
as the principal
Most microorganismsuse sulfate sulfursource and contain
reductionof sulfateto sulhde.This process of
enzyme systems for the
in Chapter 3 (see
assimilatorysulfatereduction has been discussed
as terminal electron
Fig.3.3).In sulfidefermentation,sulfateis used
formed is excreted. This process is
acceptor,and the hydrogen sulfide
thereforecalleddissimilatory sulfatereduction;it iscarriedout only by
anaerobic bacteria.On the basis of theiroxidativeabilities
strictly
the
bacteria(sulfidogenicbacteria)can be subdividedinto two
sulfate-reducing
in
groups.Representativespeciesofthesegroups are given Table 8.11.
The incomplete oxidizers, such as the Desulfovibriospecies and most
Desulfotomaculum species, have been known fora longtime;they oxidize
a number of organicacidsand alcoholsto acetate.The complete oxidizers
were recentlydiscoveredby Pfennigand co-workers. Desulfotomaculum
acetoxidanswas the first;
others,morphologically very diferent, followed.
Sulfate-reducingbacteriaare loaded with electronand hydrogencar
riers.Cytochrome c3 discoveredby Postgate,was the firstcytochrome
SulfideFermentation (Desulfurication) 261

3 3 3
 8: Bacterial
262
Fermentations

detectedin strict anaerobes. Allsulfate reducerscontain cytochromes

which are of the c-typeand/orof the b-type.In addition, menaquinone'
severalferredoxins, flavodoxin, r ubredoxin, and desulforedoxin (closelv
are
relatedto rubredoxin) present. Another remarkable constituentof
sulfatereducers isa siroheme a
protein,protein containing two tetrapyrrol
ringsystems and iron sulfurc enters. T his proteine xhibitss ulfite reductase
activityand may catalyze six-electron-transfer reactions; i t is known as
desulfoviridin
(Desulfovibrio, Desulfomonas,DesulfococCus, Desulfonema
species)or P582 (Desulfotomaculum,Desulfonema species).Firstthe
fermentation of lactateby the incompleteoxidizers willbe discussed.

A. Fermentationof lactateand sulfate

Thisfermentationcan be summarized as follows:

2CH-CHOH-COOH + 2H,0 2CH-COOH+2c0 + 8H

SO+8H S2+4H,0
Lactate oxidationto acetate proceedsvia
pyruvateand acetyl-CoA. A
flavoproteinprobably functions as in
H-acceptor the first oxidation step,
and pyruvate-ferredoxin oxidoreductase isinvolvedin acetyl-CoAforma-
tion.The finalacetate
productioniscoupled to ATP synthesis.
The 8H generatedin lactateoxidationare used to
reduce sulfateto
sulfide.Priorto the firstreductionstep,sulfateisactivatedby conversion
to
adenosine-5-phosphosulfate (APS) (Fig.8.29),and the reduction
productsare sulfite and AMP. The further
phosphorylatedcompound
PAPS, which is an intermediatein sulfatereduction
assimilatory (see
Fig.3.4),is not involvedhere. APS reductase contains
sulfur.
FAD and iron-
The reaction isreversible; in vitroAPS reduction isobserved with
reducedmethyl viologenas H-donorand
APS formationfrom sulfite and
AMP with ferricyanide as
H-acceptor.
The reductionof sulfite is catalyzed by the
reductase.Untilrecently, above-mentioned sulfite
the intermediaryformationof trithionate and
thiosulfatewas assumed, Itseems now
thatfreeintermediates do not occur
duringreductionof sulfite to sulfide.
Two aspectsof
dissimilatory reductionof sulfateto sulfide areinterest
ing: the mode of H-transferfrom lactate
oxidationreactionsto sulfate
reductionreactionsand the
of
bioenergetics sulfatereduction.
Ithas been demonstratedfor
and itselectron Desulfovibrio speciesthatone hydrogenase
acceptor,cytochrome C3, are localized in the
space. Other cytochromes, periplasmic
menaquinone, other redox carriers and lactate
dehydrogenase are situatedinthe membrane whereas all
the enzymes
pyruvateoxidation, forsulfatereduction, for
and a second
the cytoplasm.This enzyme hydrogenaseare in
localization led Peck and
assume that a "hydrogen co-workersto
cycling occursin sulfatereducers.As
in Fig.8.29,the reducing indicated
equivalents are released
by the cytoplasmic
 Sulfide
Fermentation(Desulfurication) 263

membrane

cytoplasm periplasm
2CH-CHOH-COOH

2X
2XH

2CH-CO-cOOH
4H2
2CoA

2CO, - 2Fd
4H2

2FdH,
hydrogenase
2CH,-CO-CoA So
ATP
2P
2CoA
H,O cytC3
APS
2H
2CH,-COOo Be

2P
2ADP)
AMP
2ATP 6H
so
2CH-CoOH
3H,0

ATP balance

2 lactate 2 acetatet
2C0

2ATP

so so
chemiosmotic mechanism

3ATP

Figure8.29.Pathway of dissimilatory sulfatereduction in Desulfovibrio

species
and "hydrogencycling" 1,
hypothesis. Lactatedehydrogenase, membrane-bound,
H-acceptor not known; 2, pyruvate-ferredoxinoxidoreductase; 3,phospho-
4,
transacetylase; acetate kinase;5, ATP sulfurylase;6 , pyrophosphatase; 7,
APS reductase;8, sulfite
reductase;9,cytoplasmichydrogenase; 10,periplasmie
hydrogenase.