stable carbon isotope fractionation by acetotrophic sulfur-reducing bacteria

$: Stable carbon isotope fractionation by acetotrophic sulfur-reducing bacteria$
R E S E A R C H A R T I C L E

Stable carbon isotope fractionation byacetotrophicsulfur-reducing bacteriaDennis Goevert & Ralf Conrad

Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

Correspondence: Ralf Conrad, Max Planck

Institute for Terrestrial Microbiology, Karl-von-

Frisch-Str., 35043 Marburg, Germany. Tel.:

149 6421 178801; fax: 149 6421 178809;

e-mail: [email protected]

Received 18 August 2009; revised 26 October

2009; accepted 30 October 2009.

Final version published online 27 November

2009.

DOI:10.1111/j.1574-6941.2009.00811.x

Editor: Gary King

Keywords

acetate turnover; Desulfuromonas;

Desulfurella; Hippea; acetyl-CoA pathway;

tricarboxylic acid cycle.

Abstract

Acetate is the most important intermediate in anaerobic degradation of organic

matter. The carbon isotope effects associated with the oxidation of acetate (eac)

were examined for four acetotrophic sulfur reducers, Desulfuromonas acetoxidans,

Desulfuromonas thiophila, Desulfurella acetivorans, and Hippea maritima. During

the consumption of acetate and sulfur, acetate was enriched in 13C by 11.5 and

11.2% in Desulfuromonas acetoxidans and Desulfuromonas thiophila, respectively.

By contrast, isotope fractionation in D. acetivorans and H. maritima resulted in

isotope enrichment factors of eac =� 6.3% and � 8.4%, respectively. These

sulfur-reducing bacteria all metabolize acetate via the tricarboxylic acid cycle, but

have different mechanisms for the initial activation of acetate. In Desulfuromonas

acetoxidans, acetyl-CoA is formed by succinyl-CoA : acetate-CoA-transferase, and

in D. acetivorans by acetate kinase and phosphate acetyltransferase. Hence, values

of eac seem to be characteristic for the type of activation of acetate to acetyl-CoA in

acetotrophic sulfur reducers. Summarizing eac-values in anaerobic acetotrophic

microorganisms, it appears that isotope fractionation depends on the mechanism

of acetate activation to acetyl-CoA, on the key enzyme of the acetate dissimilation

pathway, and on the bioavailability of acetate, which all have to be considered

when using d13C of acetate in environmental samples for diagnosis of the involved

microbial populations.

Introduction

Acetate is a key intermediate during the anaerobic degrada-

tion of organic matter. It can be used by different groups of

anaerobic microorganisms, such as those reducing nitrate,

iron, sulfate, protons, or producing methane. For example,

4 70% of biological methanogenesis in flooded soils (Con-

rad, 1999) and about 50% of sulfate reduction in marine

sediments (Soerensen et al., 1981) result from acetate

consumption. Besides several sulfate-reducing bacteria, a

few sulfur-reducing bacteria are also capable of oxidizing

acetate by a coupled reduction of elemental sulfur to sulfide.

However, little is known about carbon isotope fractionation

in acetate-dissimilating microorganisms, although such

knowledge may be helpful to diagnose the involvement of

particular groups of microorganisms in acetate turnover in

natural environments (Penning et al., 2006; Goevert &

Conrad, 2009).

Two main pathways for the oxidation of acetate by

anaerobic organisms are known: the acetyl-CoA/carbon

monoxide dehydrogenase pathway and the tricarboxylic

acid (TCA) cycle. Recently, it was observed that sulfate

reducers show differences in the fractionation of stable

carbon when they oxidize acetate via these different path-

ways (Goevert & Conrad, 2008). However, until now, to our

knowledge, all isolated sulfur reducers that were found to be

capable of utilizing acetate use the TCA cycle (Pfennig &

Biebl, 1976; Bonch-Osmolovskaya et al., 1990; Galushko &

Schink, 2000) for the following reaction:

CH3COO� þHþ þ 4S0 þ 2H2O! 2CO2 þ 4H2S ð1Þ

However, it was found that sulfur-reducing bacteria differ

in their biochemical activation of acetate. Desulfuromonas

acetoxidans activates acetate to acetyl-CoA via CoA transfer

FEMS Microbiol Ecol 71 (2010) 218–225c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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from succinyl-CoA (Gebhardt et al., 1985):

acetateþ succinyl-CoA! acetyl-CoAþ succinate ð2Þcatalyzed by a succinyl-CoA : acetate-CoA-transferase. In

contrast, for Desulfurella acetivorans the following reaction

was reported (Schmitz et al., 1990):

acetateþ ATP! acetyl-phosphateþ ADP ð3Þ

acetyl-phosphateþ CoASH! acetyl-CoAþ phosphate ð4Þwhere acetate is activated by two enzymes, acetate kinase

and phosphate acetyltransferase.

Therefore, we determined carbon isotope ratios for

Desulfuromonas acetoxidans and D. acetivorans to find out

whether sulfur reducers show different carbon isotope

fractionation when they activate acetate via different me-

chanisms. By studying fractionation in Desulfuromonas

thiophila (level of similarity to Desulfuromonas acetoxidans:

96.6%; Finster et al., 1997) and Hippea maritima (closely

related to D. acetivorans by approximately 88%; Miroshni-

chenko et al., 1999) our aim was to investigate whether

sulfur reducers with high 16S rRNA gene sequence similarity

express similar isotope fractionation. We hypothesize that if

this is true, the determination of carbon isotope fractiona-

tion may help to elucidate the initial activation mechanism

in acetotrophic, sulfur-reducing bacteria. Furthermore, it

might help to identify microbial groups of acetate utilizers

in natural environments by analyzing the isotopic signature

of acetate.

Materials and methods

Cultures and growth conditions

The following pure cultures were used in this study:

Desulfuromonas acetoxidans (DSM 684), Desulfuromonas

thiophila (DSM 8987), D. acetivorans (DSM 5264), and

H. maritima (DSM 10411) were obtained from the Deutsche

Sammlung von Mikroorganismen und Zellkulturen

(Braunschweig, Germany). All cultures were grown under

N2/CO2 (80 : 20) in 500-mL glass bottles (Ochs, Bovenden-

Lenglern, Germany) in bicarbonate-buffered mineral med-

ium. Pure cultures of Desulfuromonas acetoxidans were

incubated with shaking (120 r.p.m.) at 30 1C using the

original basal medium (Pfennig & Biebl, 1976). Desulfur-

omonas thiophila was also grown at 30 1C, using a modified

medium with the following composition (in g L�1 unless

otherwise noted): KH2PO4, 1.0; NH4Cl, 0.5; MgSO4 � 7H2O,

0.4; CaCl2 � 2H2O, 0.1; NaHCO3, 2.0; Na2S � 9H2O, 0.4; trace

element solution SL-10, 1.0 mL (Chin et al., 1998); vitamin

solution, 1 mL (Wolin et al., 1963); and resazurine at

0.1% w/v, 1 mL. Desulfurella acetivorans and H. maritima

were incubated without shaking at 55 1C. The medium used

for cultivation of D. acetivorans contained the following (in

g L�1 unless otherwise noted): KH2PO4, 0.33; NH4Cl, 0.33;

MgCl2 � 6H2O, 0.33; CaCl2 � 2H2O, 0.33; KCl, 0.33; NaHCO3,

2.0; Na2S � 9H2O, 0.5; trace element solution SL-10, 1.0 mL

(Chin et al., 1998); vitamin solution, 10 mL (Wolin et al.,

1963); and resazurine at 0.1% w/v, 1 mL. For growth of

H. maritima a similar medium was used, which additionally

contained 0.1 g L�1 yeast extract and 25 g L�1 NaCl and the

pH was adjusted to 6.0 with 5 M H2SO4 (Miroshnichenko

et al., 1999). All bacteria were grown with 3.8–5.3 mM

acetate as electron donor and 6.2 mM sulfur (subl., purum,

purchased from Fluka, Buchs, Switzerland) as electron

acceptor. For experiments, 10% of bacterial suspensions in

the late exponential phase were inoculated (resulting in a

final volume of 250 mL) and several samples from the

headspace and the liquid phase were removed to determine

pH, concentration of acetate, sulfide, carbon dioxide, and

carbon isotope composition of acetate and carbon dioxide.

All experiments were performed in triplicate.

Chemical and isotopic analyses

Chemical and isotopic analyses were performed as described

previously (Goevert & Conrad, 2008). Sulfide was deter-

mined photometrically after reaction with CuSO4 to form

CuS as described by Cord-Ruwisch (1985). CO2 was ana-

lyzed by GC using a flame ionization detector (Shimadzu,

Kyoto, Japan) after conversion to CH4 with a methanizer

(Ni-catalyst at 350 1C, Chrompack, Middelburg, the Nether-

lands). Stable isotope analysis of 13C/12C in gas samples was

performed using a gas chromatograph combustion isotope

ratio mass spectrometer system (Thermo Fisher Scientific,

Bremen, Germany). Isotopic measurements and quantifica-

tion of acetate were performed on an HPLC system coupled

to Finnigan LC IsoLink (Thermo Fisher Scientific).

Calculations

Fractionation factors for a reaction A ! B are defined after

Hayes (1993) as:

aA=B ¼ ðdA þ 1000Þ=ðdB þ 1000Þ ð5Þ

also expressed as e � 103 (1�a). The carbon isotope

enrichment factor associated with acetate oxidation (eac)

was calculated from the temporal change of d13C of acetate

as described by Mariotti et al. (1981) from the residual

reactant

dr ¼ dri þ e½lnð1� f Þ� ð6Þwhere dri and dr are the isotope compositions of the reactant

(acetate) at the beginning and of the residual acetate,

respectively, and f is the fractional yield of the product based

on the consumption of acetate (0o fo 1). Linear

FEMS Microbiol Ecol 71 (2010) 218–225 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

219Acetotrophic sulfur-reducing bacteria

regression of dr against ln(1� f) yields e as the slope of best-

fit lines.

As total oxidized carbon was distributed among different

carbon species, d13C of total inorganic carbon (dTIC) was

calculated by the following mass-balance equation:

dTIC ¼ Xgdg þ Xddd þ Xbdb þ Xcdc ð7Þwhere X is the mole fraction and d is the isotopic composi-

tion of the C of gaseous CO2 (g), dissolved CO2 (d), HCO3�

(b), and CO32� (c). The distribution of carbon among these

species was calculated using solubility and equilibrium

constants (Stumm & Morgan, 1995). dg was measured

directly, the remaining isotopic compositions were calcu-

lated from the relevant equilibrium isotope fractionation

factors (Deines & Langmuir, 1974; Mook et al., 1974):

dd ¼ ad=g þ dg þ ðad=g�1Þ1000 ð8Þ

db ¼ ab=g þ dg þ ðab=g � 1Þ1000 ð9Þ

dc ¼ ac=g þ dg þ ðac=g � 1Þ1000 ð10Þ

Results

Desulfuromonas acetoxidans and Desulfuromonas thiophila

were grown to study isotope fractionation of stable carbon

during acetate oxidation via the TCA cycle and activation of

acetate to acetyl-CoA via CoA transfer from succinyl-CoA.

This reaction was shown to drive acetate activation in

Desulfuromonas acetoxidans (Gebhardt et al., 1985) and is

assumed to be also active in Desulfuromonas thiophila (see

Discussion). During oxidation of acetate, both Desulfuro-

monas species converted acetate and sulfur into CO2 and

H2S, respectively, following the known stoichiometry [see

Eqn. (1); Fig. 1a and d]. Concentrations of CO2 are not

shown because the high concentrations of bicarbonate in the

medium made it impractical to measure yields of this

product. During sulfur reduction the preferred consump-

tion of [12C]acetate caused an enrichment of the heavier

isotope 13C in the remaining acetate (Fig. 1b and e). d13CCO2

[illustrated in Fig. 1 as TIC] became slightly depleted in 13C

with time but was not used for determination of isotope

fractionation because, as mentioned above, the high back-

ground of bicarbonate hindered a precise quantification of

the d13C of the newly formed TIC. Carbon isotope fractio-

nation of acetate during sulfur reduction was determined

using Eqn. (6), based on Rayleigh distillation (Fig. 1c and f

and Table 1). The carbon isotope enrichment factor for

acetate (eac) was calculated for three different phases of

acetate consumption to respect substrate limitation (Goe-

vert & Conrad, 2009): until 50% of acetate was consumed

(f-values between 0.0 and 0.5), between 50% and 80%

consumption (0.5o fo 0.8), and from 80% to maximum

consumption of acetate (0.8o fo 1.0). As carbon isotope

fractionation decreased after the first phase, e-values for the

range 0o fo 0.5 were finally used to determine and

compare isotope fractionation. Toward the end of experi-

ments and thus at lower substrate concentrations, acetate

might have become limiting and no longer fractionated

during consumption, as observed before (Kinnaman et al.,

2007; Goevert & Conrad, 2008, 2009). Such an effect of

bioavailability of substrates on stable isotope fractionation

has recently been explained theoretically for pollutant

biodegradation (Kampara et al., 2008; Thullner et al., 2008)

and shown to occur during acetoclastic methanogenesis

(Goevert & Conrad, 2009). It seems also to occur during

acetate oxidation under sulfur-reducing conditions.

In contrast to the two Desulfuromonas spp., D. acetivorans

(Schmitz et al., 1990) and apparently also H. maritima (see

Discussion) both activate acetate directly to acetyl-CoA,

catalyzed by the two enzymes acetate kinase and phosphate

acetyltransferase. During growth of D. acetivorans and H.

maritima, acetate was consumed below the detection limit

(approximately 20 mM) and was converted to CO2 (Fig. 2a

and d). When acetate was oxidized, again the preferred

consumption of light 12C-acetate caused an enrichment of

heavy 13C in the residual acetate (Fig. 2b and e), as expected

for a closed system approach. Similar to the Desulfuromonas

species, eac-values decreased with decreasing substrate con-

centrations (Fig. 2c and f and Table 1). But, interestingly,

isotope enrichment factors of acetate in D. acetivorans and

H. maritima differed from eac in Desulfuromonas spp. and

were significantly weaker (Table 1).

Discussion

Acetotrophic sulfate reducers apparently express different

carbon isotope fractionation when they use different bio-

chemical pathways for the oxidation of acetate, either the

TCA cycle or the acetyl-CoA pathway (Table 2). In the

present study, we found that carbon isotope fractionation of

acetate differs among acetotrophic sulfur-reducing bacteria

by up to 5%, even if they are using the same biochemical

pathway (TCA) for the oxidation of acetate. Thus, we

conclude that in these bacteria it is not the oxidation

pathway that determines carbon isotope fractionation as

suggested recently for sulfate-reducing bacteria (Goevert &

Conrad, 2008). Instead we propose that the initial activation

of acetate was important for the fractionation of acetate.

Whereas in D. acetivorans, acetate is activated by acetate

kinase and phosphate acetyltransferase at the expense of one

high-energy phosphate bond, in Desulfuromonas acetoxi-

dans, acetate is activated without expense of additional

energy. Acetate activation, by the latter reaction, exhibited

the stronger isotope effect during the subsequent oxidation


220 D. Goevert & R. Conrad

of acetyl-CoA in the TCA. The apparent isotope effect is

mainly dependent on the mechanism of the first irreversible

step (Northrop, 1981). In the acetotrophic sulfur reducers

this is the reaction of the citrate synthase, the key enzyme of

the TCA (Thauer, 1988). However, other reactions preced-

ing the citrate synthase may also influence the apparent

isotope effect. This has been termed ‘commitment to

catalysis’ (Northrop, 1981; Elsner et al., 2005). We may

assume that the energy-utilizing acetate kinase creates a

larger commitment to catalysis, so that the apparent isotope

effect by the citrate synthase is not as strongly expressed as

with the succinyl-CoA : acetate-CoA-transferase.

If our assumption is true, then the determination of eac

may help to identify the biochemical mechanism of acetate

activation in acetotrophic sulfur reducers. Hence, by

comparison of isotope enrichment in acetate among

different sulfur reducers, we assume that Desulfuromonas

thiophila (eac =� 11.2%) activates acetate directly via succi-

nyl-CoA : acetate-CoA transferase as in Desulfuromonas acet-

oxidans (eac =� 11.5%), and that in H. maritima (eac =

� 8.4%) this reaction is catalyzed via acetate kinase and

phosphate acetyltransferase as in D. acetivorans (eac =

� 6.3%). However, enzyme or genomic data are not avail-

able for these species.

The rates of acetate consumption were different in

the different cultures, increasing from H. maritima4D.

acetivorans4Desulfuromonas thiophila4Desulfuromonas

acetoxidans (Figs 1 and 2). However, eac was only weakly

1.5

2.0

7.10

7.151.5

2.0

7.00

7.05(d)(a)

0.0

0.5

1.0

6.95

7.00

7.05mm

ol

pH

0.0

0.5

1.0

6.85

6.90

6.95

mm

ol

pH

(e)(b)

–15

–10

–5

–18

–15

–5

0 –12(c) (f)

0 4 8 12 16 20

–25

–20

δ13C

(‰

)

Time (days)0 2 4 6 8 10

–24

–21δ13C

(‰

)

Time (days)

–20

–15

–10

δ13C

ac (

‰)

–21

–18

–15δ13

Cac

(‰

)

–3 –2 –1 0–30

–25

ln (1-f)–2.0 –1.5 –1.0 –0.5 0.0

–24

ln (1-f)

Fig. 1. Acetate oxidation by sulfur-reducing bacteria activating acetate (presumably) via succinyl-CoA : acetate-CoA-transferase. The figure shows

substance conversions and isotope fractionation during the catabolism of acetate in pure cultures of Desulfuromonas acetoxidans (a–c) and

Desulfuromonas thiophila (d–f). (a and d) Acetate consumption (’), H2S production (�), and pH (dashed line without symbols); the concentrations

are given as millimole per bottle. (b and e) Isotopic composition of acetate (’) and CO2 (B, illustrated as TIC). (c and f) Isotope enrichment in acetate

was calculated for three different phases of acetate consumption: solid lines show substrate levels between 0 and � 0.6 on the ln(1� f) scale

(corresponding to up to 50% acetate consumption), dashed lines show levels between � 0.6 and � 1.6 (50–80%), and dotted lines show levels

between � 1.6 and � 3.0 (from 80% to maximum consumption of acetate); the plots are based on Eqn. (6). Values are means� SE (n = 3).



correlated (r2 = 0.43) with these rates. The sulfur reducers D.

acetivorans and H. maritima with the acetate kinase all grow

at 55 1C, whereas the two Desulfuromonas species with the

succinyl-CoA : acetyl-CoA transferase grow at 30 1C. There-

fore, the respectively weaker or stronger isotope fractiona-

tion could also be influenced by the different growth

temperatures. However, the influence of temperature on

the kinetic isotope effect is not always obvious (Morasch

et al., 2001). We assume that the effect of the acetate-

activating reaction was more important than consumption

rate and temperature, but propose assaying more aceto-

trophic sulfur reducers at low and high growth temperatures

as they become available by isolation.

Differences in carbon isotope fractionation due to differ-

ent activation reactions of acetate have also been suggested

for the two acetoclastic, methane-producing archaeal gen-

era, Methanosarcina (Goevert & Conrad, 2009) and Metha-

nosaeta (Penning et al., 2006). These organisms have both

been shown to use the acetyl-CoA pathway to consume

acetate but eac-values differed by up to 21%, presumably

caused by the different activation mechanisms of acetate

that exist in these methanogens (Table 2). Also in these

methanogens, the apparent isotope effect was less strongly

expressed when the activation step preceding acetate clea-

vage expended more energy, i.e. resulted in a larger commit-

ment to catalysis.

Still, this does not explain why sulfate- and sulfur-

reducing bacteria express different isotope fractionation

even if they have the same pathway and the same activation

of acetate (Table 2). For example, for Desulfobacter postgatei

and Desulfobacter hydrogenophilus, which both use the

enzyme succinyl-CoA : acetate-CoA transferase and the

TCA cycle to activate and oxidize acetate, a slightly inverse

(positive) eac has been observed (Goevert & Conrad, 2008).

These differences in fractionation may be explained by the

energetics, which are completely different between sulfur

reducers and sulfate reducers and that result in the utiliza-

tion of different key enzymes for the TCA, i.e. the ATP-

citrate lyase and the citrate synthase for sulfate reducers and

sulfur reducers, respectively (Thauer, 1988). Acetate oxida-

tion in sulfur reducers such as Desulfuromonas acetoxidans

or D. acetivorans [see Eqn. (1); Pfennig & Biebl, 1976] yields

much less free energy (DG0=� 39 kJ mol�1) than acetate

oxidation in sulfate reducers (DG0=� 63 kJ mol�1 acetate).

In sulfur reducers most of the energy available from the

oxidation of 1 mol acetate with 4 mol sulfur is liberated

during the reduction of elemental sulfur to H2S rather than

during the oxidation of acetate. This was shown by Thauer

(1988) calculating the Gibbs free energy for each of the two

half reactions using the average redox potentials of NADP,

ferredoxin, menaquinone and NAD (E0=� 279 mV), of S0/

H2S (E0=� 240 mV), and of the CO2/acetate couple

(E0=� 291 mV).

CH3COO� þHþ þ 2H2O! 2CO2 þ 8½H�DG

0 ¼ �9:2 kJ mol�1 ð11Þ

8½H� þ 4S0 ! 4H2S

DG0 ¼ �29:8 kJ ð4 mol S0Þ�1 ð12Þ

In contrast, in the sulfate reducer Desulfobacter postgatei

(Widdel & Pfennig, 1981) when oxidizing 1 mol acetate with

1 mol sulfate it is the dehydrogenation of acetate [Eqn. (13)]

rather than the reduction of sulfate [SO42�/H2S:

E0=� 208 mV; Eqn. (14)] that is the major energy-yielding

reaction (Thauer, 1988). Again, DG0

for reactions (13) and

(14) were calculated from the average redox potential of the

four different electron acceptors used in the TCA cycle

(NADP, ferredoxin, menaquinone, and malate), which in

sulfate reducers (E0=� 241 mV) is supposed to be less

Table 1. Isotope enrichment factors for acetate during acetotrophic sulfur reduction by Desulfuromonas acetoxidans, Desulfuromonas thiophila,

Desulfurella acetivorans, and Hippea maritima�

Sulfur-reducing acetotroph f Acetate concentration (mM) eac (%) r2w

Desulfuromonas acetoxidans 0.0–0.5 3.8–1.9 � 11.45� 0.21 0.949

0.5–0.8 1.9–0.8 � 10.14� 0.61 0.997

0.8–1.0 0.8–0.0 � 2.85� 1.87 0.913

Desulfuromonas thiophila 0.0–0.5 5.3–2.7 � 11.19� 0.43 0.989

0.5–0.8 2.1–1.1 � 4.76� 0.24 0.999

0.8–1.0 1.1–0.0 ND

Desulfurella acetivorans 0.0–0.5 4.2–2.1 � 6.28� 1.30 0.910

0.5–0.8 2.1–0.8 � 3.50� 0.62 0.990

0.8–1.0 0.8–0.0 � 0.63 1

Hippea maritima 0.0–0.5 3.8–1.9 � 8.38� 1.04 0.978

0.5–0.9 1.9–0.4 � 1.20 1

0.9–1.0 0.4–0.0 � 0.52 1

�Values are means of triplicates.wCoefficient of determination. ND, not determined.



negative than in the sulfur reducers:

CH3COO� þHþ þ 2H2O! 2CO2 þ 8½H�DG

0 ¼ �38:8 kJ mol�1 ð13Þ

8½H� þ SO2�4 þ 2Hþ ! H2Sþ 4H2O

DG0 ¼ �24:2 kJ mol�1 ð14Þ

Here, 2 mol of ATP are required for the activation of

sulfate. On the other hand, 1 mol of ATP is formed during

dehydrogenation of acetate, as it was found that the

synthesis of citrate from oxaloacetate and acetyl-CoA in

Desulfobacter postgatei is catalyzed by an ATP-citrate lyase

rather than by citrate synthase as in Desulfuromonas acetox-

idans (Moller et al., 1987; Schauder et al., 1987). The ATP-

citrate lyase in sulfate reducers with TCA probably results in

a smaller apparent isotope fractionation during acetate

consumption than the citrate synthase in sulfur reducers

with TCA (Table 2).

The energetics are also different between acetotrophic

Methanosarcina species (DG0=� 36 kJ mol�1 acetate) and

Desulfobacca acetoxidans (DG0=� 63 kJ mol�1 acetate)

(a) (d)

1.5

2.0

6.1

6.22.0 7.25

(b) (e)–18

–150.0

0.5

1.0

5.8

5.9

6.0

mm

ol

pH

–21

–18

0.0

0.5

1.0

1.5

7.05

7.10

7.15

7.20

mm

ol

pH(c)

–15(f)

–18

0 1 2 3 4 5 6 7

–27

–24

–21

δ13C

(‰

)

Time (days)0 1 2 3

–27

–24δ13C

(‰

)

Time (days)

–21

–18

δ13C

ac (

‰)

–24

–21

δ13C

ac (

‰)

–3 –2 –1 0–27

–24

ln (1-f)–4 –3 –2 –1 0

–27

ln (1-f)

Fig. 2. Acetate oxidation by sulfur-reducing bacteria activating acetate (presumably) using acetate kinase and phosphate acetyltransferase. The figure

shows substance conversions and isotope fractionation during the catabolism of acetate in pure cultures of Desulfurella acetivorans (a–c) and Hippea

maritima (d–f). (a and d) Acetate consumption (’), H2S production (�), and pH (dashed line without symbols); the concentrations are given as millimole

per bottle. (b and e) Isotopic composition of acetate (’) and CO2 (B, illustrated as TIC). (c and f) Isotope enrichment in acetate was calculated for three

different phases of acetate consumption: solid lines show substrate levels between 0 and �0.6 on the ln(1� f) scale (corresponding to up to 50%

acetate consumption), dashed lines show levels between � 0.6 and � 1.6 (or the next data point; 50 to 80%), and dotted lines show levels between

� 1.6 and � 3.5 (from 80% to maximum consumption of acetate); the plots are based on Eqn. (6). Values are means� SE (n = 3).



despite use of the same mechanism of acetate activation

(acetate kinase) and the same dissimilatory pathway (acetyl-

CoA pathway) (Table 2). The Methanosarcina species with

less energy available than Desulfobacca acetoxidans showed a

stronger fractionation. The catalytic basis of the different

isotope fractionation is unclear at present. However, we

hypothesize that the commitment for catalysis was larger in

Desulfobacca than in Methanosarcina spp.

In conclusion, we propose that closely related aceto-

trophic sulfur reducers (based on high 16S rRNA gene

sequence similarities) use the same mechanism for the

activation of acetate to acetyl-CoA and thus express similar

isotope fractionation of stable carbon. In general, the

apparent isotope fractionation in anaerobic acetotrophs is

probably affected not only by the acetate dissimilation

pathway and its key enzymes (citrate synthase, ATP-citrate

lyase, acetyl-CoA synthase complex) but also by the preced-

ing acetate activating enzymes (acetate kinase, acetyl-CoA

synthetase, succinyl-CoA : acetyl-CoA transferase) creating a

particular commitment of catalysis. These effects are fully

expressed only if acetate is not limiting. However, if the

concentration of acetate is so low that it is not fully available

to the microorganisms, the apparent stable isotope fractio-

nation is decreased (Thullner et al., 2008; Goevert & Con-

rad, 2009). Hence, quite a number of factors have to be

considered when acetate fractionation data collected in

nature are to be used for diagnosis of the operation of a

particular type of acetate dissimilation pathway or acetate-

dissimilating microbial community.

Acknowledgements

This work was supported by the Fonds der Chemischen

Industrie, Germany. We thank Peter Claus for excellent

technical assistance and Rudolf Thauer for helpful discus-

sions. We also thank two anonymous reviewers for helpful

comments.

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stable carbon isotope fractionation by acetotrophic sulfur-reducing bacteria

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