First publ. in: Archives of microbiology 151 (1989), 2, pp.

126 - 132

Anaerobic degradation of isobutyrate by methanogenic enrichment

cultures and by a Desuifococcus multivorans strain
Marion Stieb * and Bernhard Sehink**

Fakult/it fiir Biologic, Universit/it Konstanz, Postfach 5560, D-7750 Konstanz, Federal Republic of Germany
Abstract. Methanogenic enrichment cultures with isobuty-

rate as sole source of carbon and energy were inoculated with

sediment and sludge samples from freshwater and marine
origin. Over more than 20 transfers, these cultures fermented
2 tool isobutyrate with 1 mol CO2 via an intermediate formarion of n-butyrate to 4 mol acetate and I tool CH4. The
primary isobutyrate-fermenting bacteria could not be
purified. From one of the marine enrichment cultures, a
sulfate-reducing bacterium was isolated which oxidized
isobutyrate with sulfate completely to COz. Based on its
physiological and morphological properties, this strain was
assigned to the known species Desulfococcus multivorans. It
also oxidized many other fatty acids without significant
release of short-chain intermediates. The enzymes involved
in isobutyrate degradation by this bacterium were assayed
in cell-free extracts. The results indicate that isobutyrate is
activated to its CoA derivative and oxidized via methylmalonate semialdehyde to propionyl-CoA. Propionyl-CoA
is further converted via the methylmalonyl-CoA pathway to
acetyl-CoA which is finally cleaved by the CO-dehydrogenase system. It is evident that this is not the pathway used
by the fermenting bacteria prevailing in the methanogenic
enrichment cultures. These results are discussed on the basis
of energetical considerations.
Key words: Fatty acid degradation -

Syntrophic associations - Isobutyrate - Sulfate-reducing bacteria Isobutyrate-butyrate isomerization - CO-Dehydrogenase

the cultivation of ruminal anaerobes (Tanner and Wolfe

1988). For other anoxic ecosystems, such as marine or freshwater sediments, complete degradation has to be postulated.
In marine sediments rich in organic compounds, a constant
pool size of e.g. 0 . 5 - 6 . 0 gM isobutyrate is maintained
(Sansone and Martens 1981, 1982). Methanogenic degradation of isobutyrate via intermediate formation of acetate
was observed in anaerobic digestor contents (Zinder et al.
1984; Tholozan et al. 1988).
Some s.ulfate-reducing bacteria were recently isolated
which are able to oxidize branched-chain fattyacids either
incompletely to acetate residues or completely to carbon
dioxide with concomitant formation of sulfide (Widdel and
Pfennig 1984; Widdel 1988). In the absence of sulfate, 2methylvalerate is fermented by syntrophic methanogenic associations to acetate, propionate, and methane (Stieb and
Schink 1985), and 3-methylvalerate is converted with COz
to hydrogen and 3 tool of acetate by a highly specialized,
obligately syntrophic bacterium (Stieb and Schink 1986).
In the present communication, complete degradation of
isobutyrate in mesophilic methanogenic enrichment cultures
is documented. It turns out that the pathway of isobutyrate
degradation in these cultures differs basically from that used
by a sulfate-reducing bacterium isolated from the same cultures.

Materials and methods

Sources o f organisms

Branched-chain fatty acids such as isobutyrate, 2-methylbutyrate, and 3-methylbutyrate, play an important role as
intermediates in the anaerobic degradation of lipids and
amino acids. Anaerobic bacteria excrete these fatty acids
during fermentative degradation of valine, isoleucine, and
leucine (Barker 1981; Massey et al. 1976; Allison 1978;
Harwood and Canale-Parola 1981). In the rumen of cattle,
branched-chain fatty acids can be used again for reductive
synthesis of the respective amino acids by other microorganisms (Allison and Bryant 1963; Allison 1969), and they proved repeatedly as growth-stimulating medium additions in
* Present address: Dornier System GmbH, Space Development

Division, D-7990 Friedrichshafen 1, FRG

** Present address and address for offprint requests: Lehrstuhl

Mikrobiologie I, Auf der Morgenstelle 28, D-7400 Tfibingen, FRG

Enrichment cultures with 10 mM isobutyrate in either freshwater of saltwater medium were inoculated with several
different sediment and sludge samples of freshwater and
marine origin. Strain Gralbul was isolated from a marine
enrichment culture inoculated with sediment of Canal
Grande, Venice, Italy, and compared with Desulfococcus
multivorans strain lbel, DSM 2059, which was kindly provided by F. Widdel.
Cultivation and isolation

All procedures for cultivation as well as for gas chromatographic determination of fatty acids and methane were
essentially as previously described (Widdel and Pfennig
1981 ; Schink and Pfennig 1982; Stieb and Schink 1984). The
mineral medium for enrichment and further cultivation was

Konstanzer Online-Publikations-System (KOPS)

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127
carbonate-buffered and sulfide-reduced, and contained the
trace element solution SL 10 (Widdel et al. 1983). The pH
was adjusted to 7.2 - 7.4. Freshwater medium contained 1 g
NaC1 and 0.4 g MgCI2 6 H20, saltwater medium 20.0 g
NaC1 and 3.0 g MgC12 x 6 H20 per 1. Isolation of defined
mixed cultures was tried by repeated application of the agar
dilution method (Pfennig 1978) in the presence and absence
of Desulfovibrio vulgaris strains Marburg and E 70 (marine
isolate), or Methanospirillum hungatei strain M l h as hydrogen scavenger. All growth experiments were carried out at
28~
Growth yields and substrate conversion stoichiometries were determined in 23 ml screw cap tubes which
could be inserted directly into a Bausch and Lomb
Spectronic 20 spectrophotometer for density measurements
at 440 nm wavelength. With methanogenic enrichment cultures, product formation was measured in serum bottles
sealed with butyl rubber septa. All growth experiments were
carried out in duplicates which differed in their results by
10% at maximum.
The G r a m type was determined according to Gregersen
(1978) and to Magee et al. (1975). Escherichia coli and
Acetobacterium woodii were used as controls.

to Brandis-Heep et at. (1983), succinate dehydrogenase, realate dehydrogenase, and fumarate hydratase according to
Stams et al. (1984). Isocitrate dehydrogenase was determined
as described by Bernt and Bergmeyer (1974), and
methylmalonyl-CoA decarboxylase after Hilpert et al.
(1984). Formate acetyl transferase activity was determined
as oxaloacetate-dependent citrate synthesis or acetyl-CoAdependent pyruvate formation according to Knappe et al.
(1974). Acetate kinase and phosphate acetyl transferase were
measured by standard procedures (Bergmeyer 1974). Protein
was quantified by a microbiuret method (Kuenen and
Veldkamp 1972). Sulfide was measured by the methylene
blue method (Cline 1969).

Chemicals
All chemicals used were of analytical grade quality and
obtained from Merck, Darmstadt, FRG, or Fluka, NeuUlm, FRG. Biochemicals and enzymes were purchased from
Boehringer, Mannheim, FRG, and Sigma Chemical Co.,
Mfinchen, FRG.

Results

Enzyme assays

Enrichment cultures with isobutyrate

Cells of Desulfococcus multivorans strain G r a I b u l were

grown with either 5 mM propionate or 5 mM isobutyrate in
the presence of 20 mM sulfate, and harvested in the late
exponential growth phase. For enzyme assays, either French
pressure cell extracts or cell suspensions treated with cetyl
trimethyl ammonium bromide were used. Details of the
preparation procedure were described by Schink et al.
(1987). Care was taken to avoid any exposure of the cell
material to air. All enzyme assays were carried out in a Zeiss
PM 4 spectrophotometer in anoxic 1 ml cuvettes at 30 ~C.
Acyl-CoA dehydrogenases were determined following
dichlorophenol indophenol (DCPIP) reduction with butyryl-CoA or isobutyryl-CoA as substrates (Green et al. 1954).
Butyryl-CoA synthetase and isobutyryl-CoA synthetase
were measured as coupled reactions with the respective acylCoA dehydrogenases as DCPIP reduction in the presence
of 20 mM free fatty acid and 2 mM ATP; the reaction was
started by addition of 2 IJ 40 mM CoASH. SuccinylCoA: fatty acid CoA transferases were measured according
to Brandis-Heep et al. (1983) or to Hilpert et al. (1984).
Enoyl-CoA hydratase was determined with 3-methylcrotonyl-CoA as substrate (Moskowitz and Merrick 1969).
3-Hydroxyisobutyryl-CoA hydrolase was determined with
3-methylcrotonyl-CoA as substrate (Rendina and Coon
1957); 3-hydroxyisobutyrate dehydrogenase according to
Robinson and Coon (1957). The 3-hydroxyisobutyrate used
was prepared from the commercially available methylester
by acid hydrolysis (10rain boiling in 0 . 5 M HC1).
Methylmalonate semialdehyde dehydrogenase was assayed
with benzylviologen (BV) or N A D + and either propionaldehyde or acetaldehyde as substrate; pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, formate dehydrogenase, carbon monoxide dehydrogenase and
hydrogenase were determined with the same acceptors
(Schink 1985a). Methylmalonyl-CoA:pyruvate transcarboxylase was determined according to Schink et al. (1987),
oxaloacetate decarboxylase according to Scrutton et al.
(1979), succinate thiokinase and citrate synthase according

50 ml-enrichment cultures with freshwater or saltwater medium and 10 mM isobutyrate as sole source of carbon and
energy were inoculated with sediment and sludge samples
from various sources. Gas formation was observed after 1 9 weeks of incubation, and ceased after further 3 - 4 weeks.
Transfers were made with 5 ml culture fluid, and care was
taken that also some sediment particles were transferred.
This proved to be essential to secure rapid resumption of
gas formation in the subcultures. After 3 - 4 transfers, the
cultures did not contain sediment particles any more, and
purification of the primary isobutyrate-fermenting bacteria
was tried in agar dilution cultures with and without Desulfovibrio vulgaris or Methanospirillum hungatei as hydrogen
scavengers. However, in spite of numerous efforts, no defined cocultures could be isolated from the grown colonies.
Figure 1 shows the cell types present in a freshwater enrichment culture originally inoculated with sewage sludge from
the municipal sewage plant in Konstanz, after 10 subsequent
transfers. At this stage, the cultures were nearly free of
acetate-cleaving Methanothrix soehngenii cells. Motile,
slightly curved, rod-shaped, fluorescing bacteria similar to
M. hungatei predominated together with banana-like curved
rods that formed spore-like inclusions in older cultures.
Some short rods as well as thin curved rods were also present
in significant numbers.
Substrate utilization, growth, and product formation by
this enrichment culture are shown in Fig. 2. Growth and
acetate formation follow nearly exponential kinetics with a
doubling time of about 3 days. Butyrate is formed as an
intermediate product up to a concentration of 1.2 raM, and
disappears in the late logarithmic growth phase. Acetate and
methane are the final products formed. Degradation
of 300 lxmol isobutyrate yielded 585 I~mol acetate and
132 gmol methane. This corresponds to an electron recovery
of 95.6% ; the missing electrons were probably consumed in
cell material synthesis. Similar results were obtained with
three further freshwater and saltwater enrichment cultures,
however, growth in these cultures was slower.

128

Fig. 1. Phase contrast photomicrograph of the isobutyrate enrichment culture WoIbu after 12 transfers. Bar equals 10 gm

.A-

Both G r a m staining as well as G r a m typing by the K O H method characterized this strain to be Gram-negative. No
indication of spore formation was found. Comparison with
the described Desulfococcus multivorans strain lbel
suggested that this new strain belonged to the same species.
The stoichiometry of substrate utilization as well as product
and cell material formation by this strain with four different
fatty acids as substrate are shown in Table 1. Growth with
acetate was extremely slow and the yields small, but reproducible results could be obtained after 4 - 5 weeks of incubation. Growth with the other three fatty acids was by far
faster (td = 3 - 4 days) and yielded much more cell material.
Growth was stimulated significantly by addition of small
amounts of dithionite. From comparison with the four substrates at various concentrations it appeared that the yields
decreased with hydrogen sulfide concentrations higher than
about 8 mM. Molar growth yields with butyrate and
isobutyrate were nearly identical.
During growth with butyrate and isobutyrate at 5 m M
concentration, small quantities of acetate were released
(Table 1). This acetate excretion could not be significantly
enhanced by limitation of sulfate supply. Cocultures of this
strain with M. hungatei in the absence of sulfate did not
degrade isobutyrate or other fatty acids.

Enzymes involved in isobutyrate oxidation

~oo

o,2
D--J3

20o~-/

[]

~ B ~
9/~<~o~

cO

o~

o,1
A~ z ~ A

o,l,~--'<*,t
0

ut,=~'~
4

~o

'

12

16

"time[d]

Fig. 2. Time course of isobutyrate fermentation by the enrichment

culture WoIbu after 12 transfers. (9 Isobutyrate, ( l ) butyrate,
( 9 acetate, (A) methane,([]) optical density at 600 nm wavelength

Isolation and characterization of strain Gralbul

In one attempt to isolate a marine syntrophic isobutyrateoxidizing bacterium from Canal Grande sediment in the
presence of D. vulgaris and sulfate, a coccoid bacterium was
isolated which was independent of hydrogen scavengers. It
oxidized isobutyrate in pure culture with sulfate as electron
acceptor. Further substrate utilization tests showed that it
also grew with straight-chain fatty acids of 1 - 1 8 carbon
atoms including propionate, 2-methylbutyrate, 3-methylbutyrate, ethanol, propanol, butanol, fumarate, succinate, salicylate, phenylacetate, phenylpropionate, benzoate, and
cyclohexane carboxylate. These substrates were all completely oxidized to COz with concomitant sulfide formation.
Cells were round, 1 . 3 - 2.5 lam in diameter, nonmotile, and
tended to stick together in pairs or clumps (Fig. 3a). In
Indian ink preparations, capsulae became visible (Fig. 3 b).

Enzymes involved in the degradation of fatty acids were

assayed in cell-free extracts of isobutyrate- and propionategrown cells. The results are shown in Table 2. Most of the
enzymes involved in an assumed/?-oxidation of isobutyrate
after activation via a CoA transferase reaction were detected
at moderate activities sufficient to allow growth at the rates
observed. 3-Hydroxyisobutyrate dehydrogenase and methylmalonate-semialdehyde dehydrogenase were both present in isobutyrate-grown cells at significantly higher activities than in propionate-grown cells. Most of the other enzymes were found at nearly equal activities in both cell
preparations indicating that they were involved in energy
metabolism on both substrates to a similar extent. Some of
the enzymes of the tricarboxylic acid cycle were found at
very low activity, however, no 2-oxoglutarate dehydrogenase
could be detected. Instead, high activities of benzyl viologen
(BV)-dependent carbon monoxide dehydrogenase was present indicating that acetyl-CoA was oxidized through this
enzyme system, in combination with formate dehydrogenase
(BV dependent). Pyruvate synthase (pyruvate: ferredoxin
oxidoreductase) was strictly dependent on the presence of
coenzyme A. Hydrogenase activity was low; the bacterium
was unable to grown with hydrogen aselectron donor.

Discussion

Methanogenic degradation of isobutyrate

The stoichiometry of isobutyrate degradation in methanogenic cultures has not yet been documented so far. From the
results presented in this publication it appears that transformation of isobutyrate to methane and CO2 by a so far
non-defined microbial community proceeds via an intermediate formation of n-butyrate and 2 tool of acetate per
tool isobutyrate. The bacteria involved in the conversion of
isobutyrate to butyrate and in the further fermentation of
butyrate to acetate and hydrogen could not be isolated or

129

Fig. 3a, b
Phase contrast photomicrograph of
Desulfococcus multivorans strains
GraIbul. a Free cell suspension, b Indian Ink preparation. Bar equals 10 gm
for both panels
Table 1. Substrate utilization stoichiometry and growth yields of Desulfococcus multivorans strain GraIbul in batch cultures
Substrate

Acetate
Acetate
Acetate

Conc.
(mM)

net OD

Dry weight
(mg/l) a

Substr. assim.
(mM) b

Products (mM)
H2S

acetate

Electron
recovery
(%)

Yield
(g/too0

5
t0
15

0.05
0.10
0.12

10.5
21.0
25.2

0.204
0.41
0.52

5.0
9.0
12.7

< 0.01
< 0.01
< 0.01

102
94
89

2.1
2.1
(1.7)

Propionate
Propionate

2
5

0.10
0.23

21.0
48.3

0.25
0.57

3.3
7.2

< 0.01
< 0.01

101
94

10.5
(9.7)

Butyrate
Butyrate

2
5

0.17
0.30

35.7
63.0

0.295
0.52

4.4
9.2

< 0.01
0.35

103
85

17.8
(12.8)

Isobutyrate
Isobutyrate

2
5

0.18
0.30

37.9
63.0

0.313
0.52

4.0
9. t

< 0.01
0.49

96
86

18.9
(12.8)

Growth experiments were carried out in 23 ml screw cap tubes with mineral medium containing the respective substrate and a trace of
sodium dithionite. Tubes were incubated for up to 32 days and checked at intervals of 2 days
a Cell dry weights were calculated by comparison of OD values with values obtained in 2 replicate 500 ml cultures grown with isobutyrate
under the same conditions in which dry weights were determined gravimetrically
b Assimilation of substrate into cell material was calculated using the formula (C4HvO3) for cell material

identified. So, the question remains open whether both reactions are carried out by the same bacterium or by two different ones. In the latter case, the first bacterium would not get
any energy from this conversion reaction, and it would be
hard to explain how it could be transferred in the enrichment
cultures through numerous subcultivations. It appears more
probable that isobutyrate-butyrate isomerization and the floxidation of butyrate are being carried out by the same
bacterium, but in this case we cannot explain why this bacterium could not be isolated yet. Reversible isomerization
o f isobutyrate to butyrate and vice versa has also been observed in enrichment cultures from mesophilic digestor contents (Tholozan et al. 1988). N M R experiments with this
culture suggest that this conversion occurs by migration of
the carboxylic group, e.g. by a coenzyme B12-dependent
reaction. A similar interconversion o f butyrate to
isobutyrate was observed in freshwater sediment samples
incubated under hydrogen (Lovley and Klug 1982). Degradation o f isobutyrate by a defined culture of a syntrophic
fatty acid-oxidizing, straight rod-shaped Clostridium sp. as
claimed in the isolation report of this strain (Shelton and
Tiedje 1984) could not be reproduced (Tiedje, unpublished
work). This isolate probably has to be assigned to the recently described Clostridium bryantii (Stieb and Schink
1985).

Isobutyrate oxidation by Desulfococcus multivorans strain

Gralbul
A sulfate-reducing bacterium was isolated from one of the
marine enrichment cultures with isobutyrate in the presence
of sulfate. This strain was assigned to the known species
Desulfococcus rnultivorans (Widdel and Pfennig 1984;
Widdel 1988) on the basis o f its morphological and physiological properties. It did not degrade isobutyrate in the absence o f sulfate in syntrophic association with Methanospirillum hungatei, as this is true as well for all fatty aciddegrading sulfate reducers if fatty acids are provided as
substrates (Widdel 1988). It can be ruled out, therefore, that
this strain cooperated with hydrogen-scavenging methanogens in syntrophic isobutyrate oxidation; moreover the
strain was not present in the enrichment cultures in significant numbers. Transfer and survival of this strain in our
sulfate-free enrichment culture over numerous subcultivations can be explained by its ability to reduce also
thiosulfate; this is often present in small but significant
amounts as contamination of the commercially available
sodium sulfide preparations used as reductant.
The pathway of isobutyrate oxidation by this strain has
been studied on the basis of enzyme measurements in cellfree preparations. The results can be interpreted by the

130
Table 2. Specific activities (gmol 9rain-1, mg protein 1) of enzymes detected in cells of Desulfococcus multivoransstrain GraIbul
Enzyme

Butyryl-CoA synthetase
Succinyl-CoA:proprionate CoA transferase
Succinyl-CoA:isobutyrate CoA transferase
Butyryl-CoA dehydrogenase
Enoyl-CoA hydratase
3-Hydroxyisobutyryl-CoA hydrolase
3-Hydroxyisobutyrate dehydrogenase (NAD)
Methylmalonate-semialdehyde dehydrogenase (BV)a
Methylmalonyl-CoA :pyruvate transcarboxylase
Succinate dehydrogenase
Fumarate hydratase
Malate dehydrogenase (NAD)
Formate acetyl transferase (?)
Pyruvate synthase
Formate dehydrogenase (BV)
Carbon monoxide dehydrogenase (BV)
Hydrogenase (BV)

EC number

6.2.1.2
2.8.3.1
2.8.3.1 (?)
1.3.99.2
4.2.1.17
3.1.2.4
1.1.1.31
1.2.1.27 (?)
2.1.3.1
1.3.99.1
4.2.1.2
1.1.1.37
2.3.1.54
1.2.7.1
1.2.1.2
?
1.18.99

Activity in cells grown with

Isobutyrate

Propionate

0.03
0.13
0.12
0.013
0.28
10.1
0.21
0.17
0.30
0.12
0.12
3.2
0.07
0.44
0.61

n.d.
0.11
0.13
n.d.
n.d.
n.d.
0.05
0.062
0.37
0.11
0.18
!.2
0.11
0.34
O.69

1.25

1.25

0.011

0.010

n.d., means not determined; BV, benzylviologen

a Measured with propionaldehyde as substrate. Measurement with acetaldehyde as substrate gave about tenfold lower activities
Enzymes not detected (activities < 0.01 gmol 9min-1 . g protein-1): Isobutyryl-CoA synthetase, succinyl-CoA:acetate CoA transferase,
succinate thiokinase, methylmalonate-semialdehyde dehydrogenase (NAD), methylmalonyl-CoA decarboxylase, oxaloacetate decarboxylase, pyruvate dehydrogenase (NAD), citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase (NAD, BV), phosphate acetyltransferase, acetate kinase

scheme given in Fig. 4. The degradation pathway basically

follows the one found in aerobic oxidation of valine and
isobutyrate by pig kidney tissue (Robinson et al. 1957) and
by aerobic bacteria (Sokatch 1966; Sokatch et al. 1968;
Massey et al. 1976). Isobutyrate is activated to its CoA
derivative, probably by a coenzyme A transferase exchanging with succinyl-CoA. Oxidation in two steps and hydrolysis leads to methylmalonic semialdehyde and via a coenzyme
A-dependent oxidation and decarboxylation to propionyl
CoA. Oxidation of propionyl CoA follows the methylmalonyl-CoA pathway; carboxyl transfer is catalyzed by
a methylmalonyl-CoA:pyruvate transcarboxylase. Cleavage
of pyruvate to acetyl-CoA and a C1 moiety was catalyzed
by pyruvate synthase (pyruvate ferredoxin oxidoreductase)
as this is usual among the strict anaerobes. The enzyme was
highly dependent on coenzyme A in the assay buffer. A slight
activity of formate acetyltransferase (pyruvate formate
lyase) could be detected which, however, could also be a
side-effect of pyruvate synthase. Indications of occurrence
of formate acetyl transferase were recently found in a strictly
anaerobic fermenting bacterium, Ilyobacter polytropus
(Stieb ans Schink 1984). Further oxidation of acetyl CoA is
catalyzed by the CO dehydrogenase system which appears
to predominate among the acetate-oxidizing sulfate reducers
(Schauder et al. 1986). Propionate or acetate appear to enter
the described pathway at the sites indicated via activation
by
CoA
transferases
or
the
acetate
kinasephosphotransacetylase system.

Energetics of methanogenic isobutyrate oxidation

The pathway of isobutyrate degradation by D. multivorans
as lined out above cannot be the main pathway of
isobutyrate degradation in the methanogenic enrichment
culture, since the former only forms one acetate residue

instead of two. Obviously, the methanogenlc coculture predominantly uses a fi-oxidation to form two acetate residues
after initial conversion of isobutyrate to butyrate. The reason for using a different pathway may be due to energetical
problems.
All syntrophic fatty acid oxidations have to cope with
the same energetical problems of electron release in the
form of free hydrogen. These reactions are endergonic under
standard conditions [all calculations after Thauer et al. 1977;
syntrophic acetate oxidation was recently described (Zinder
and Koch 1984; Lee and Zinder 1988)]:
Butyrate- + 2 H20 ~ 2 Acetate- + H + + 2 H2
AG'o = + 48.3 kJ per tool butyrate
= + 24.2 kJ per mol U2~
Propionate- + 2 H 2 0 -* Acetate- + CO2 + 3 H2
AG'o = + 71.7 kJ per mol propionate
= + 23.9 kJ per mol H2 9
Acetate- + H + + 2 H 2 0 ~ 2 CO2 + 4H2
AGo = + 94.9 kJ per mol acetate
= + 23.7 kJ per mol H2 9
It appears that the amount of free energy required for transfer of 1 mol hydrogen under standard conditions is the same
in all cases, which means that transfer can occur in all instances at the same low hydrogen partial pressures. The
value is also the same for an assumed syntrophic oxidation
of isobutyrate to one acetate and two CO2:
Isobutyrate- + 4 H20 ~ Acetate- + 2 CO2 + 6 H2
AGo = + 143.2 kJ per mol isobutyrate
= + 23.9 kJ per mol H2 9
The problem is therefore not simply one of total conversion
energetics but rather one of the electron transfer mechanism.

131
CH3
I
..~0
H3C-CH-C'o-

(~A
CO2 <7"--- [COl~

J"
"C)P

C~ SH ("

2[HI

$
~,0
H3C-CH-C" S-CoA
I

a, ,I, a,
6[H]

(g)=

I~

CH3-C~'0
"S-CoA
@2ZH]
C02

CH3

2 EH~

~)A
X-CHa ~ . ~ ' ~ . ~ - > CO2

CH3-C-COOCH3

[CO2]

.~eo'-<~
H20

"| ~

-~176

Acknowledgements. The authors are indebted to Prof. N. Pfennig

and Dr. F. Widdel for helpful discussions throughout this study.

Financial support by the Deutsche Forschungsgemeinschaft is
gratefully acknowledged.

if-

HO CH3
I

H2C-CH-C~S - C o A
"(~

-OOC-CH2-CHOH-CO0-

CoA-SH

"~) ;
H20
-OOC-CH :CH - C(DO-

HO CH

t ~ 3 0
H2C-CH-CCo_
/

A~) p

2 [H) ~ .

z [H;

!
.j.O

l~C~CH~ICH

[H]

~C~

o>,._c:O_
2[H,

~,A

CoA_ O:C-CH2-CH2-C::_

c%
CH3
G~

0~.

T@
[COC

H
)

(isobutyryl C o A dehydrogenase and succinate dehydrogenase), however, could form only one ATP by substrate-linked
p h o s p h o r y l a t i o n (acetate kinase). This one ATP cannot
drive the reversed electron t r a n s p o r t of two electron pairs
and still leave an ATP fraction for bacterial growth. This
m a y be the reason why the syntrophic methanogenic culture
isomerizes isobutyrate to butyrate and oxidizes it to two
acetate residues.

O~,,C_C[_C r 0
CoA-S~ I ~0CH3

CHa-CH2-COO-

Fig. 4. Suggested pathway of isobutyrate oxidation by Desulfococcus multivorans strain GraIbul. Numbers in circles refer to
the following enzymes: 1 Succinyl-CoA:acid CoA transferase,
2 butyryl CoA dehydrogenase, 3 enoyl CoA hydratase, 4 3-hydroxyisobutyryl CoA hydrolase, 5 3-hydroxyisobutyrate dehydrogenase, 6 methylmalonate semialdehyde dehydrogenase, 7 methylmalonyl-CoA:pyruvate transcarboxylase, 8methylmalonyl CoA
mutase, 9 succinate dehydrogenase, 10 fumarate hydrase, 11 malate
dehydrogenase, 12 pyruvate synthase, 13 carbon monoxide dehydrogenase, 14 formate dehydrogenase. Enzymes measured in the
present study are marked (A)

D u r i n g fatty acid oxidation, electrons arise which are released at potentials rather high c o m p a r e d to that o f free
hydrogen (E'o = - 4 2 0 mV), e.g. in the succinate dehydrogenase reaction (E'o = - 3 0 mV) or in the butyryl C o A
dehydrogenase reaction (E; = - 1 2 5 mV; G u s t a f s o n et al.
1986). It has been hypothesized that these electrons require
a p r o t o n motive force-dependent reversed electron transfer
which uses up to 2/3 o f one ATP (Thauer and Morris 1984;
Schink and Thauer 1988). D u r i n g syntrophic butyrate and
p r o p i o n a t e degradation, always one pair o f electrons is released at such a high redox potential, and one ATP is being
formed by substrate-linked p h o s p h o r y t a t i o n in the acetate
kinase reaction (Wofford et al. 1986; K o c h et al. 1983;
Schink 1985b; Schink, unpublished results), thus leaving
a b o u t 1/3 o f an ATP for growth o f the respective bacteria.
Syntrophic oxidation o f isobutyrate via the D. multivorans
p a t h w a y to one acetate and two CO2 would release two
electron pairs at this unfavourable redox potential

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