Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H₂ and CO₂

Abstract

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In Acetobacterium woodii the isotope fractionation factor (ε) for ¹³C and ¹²C has previously been reported to be ε = −58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H₂ and CO₂. The average fractionation factor was ε_TIC = −57.2‰ for utilization of total inorganic carbon and ε_acetate = −54.6‰ for the production of acetate. The strongest fractionation was found for Sporomusa sphaeroides (ε_TIC = −68.3‰), the lowest fractionation for Morella thermoacetica (ε_TIC = −38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of Thermoanaerobacter kivui. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.

INTRODUCTION

Acetate is a central intermediate in the anaerobic degradation of organic matter and originates either from fermentation of organic compounds or from reduction of CO₂ with H₂ via the acetyl coenzyme A (acetyl-CoA) pathway (acetogenesis). The produced acetate serves as an important substrate for other microbes, e.g., for acetoclastic methanogens. The pathways involved in acetate turnover can be differentiated if the fractionation factors (ε) of the individual processes (e.g., fermentation, acetogenesis, and methanogenesis) are known (1). The fractionation factor can be quantified from the natural abundance of ¹³C in acetate, organic carbon, inorganic carbon, and methane, which can all be measured in environmental samples.

In anoxic environments and pure cultures of anaerobic microorganisms mainly the acetate degradation processes (methanogenesis and sulfate or sulfur reduction) have been investigated for the isotopic fractionation factors (2–10). The production of acetate, however, is less well characterized. In general it has been reported that fermentation exhibits only a weak (<5‰) fractionation (11–13). On the other hand, acetate production via chemolithotrophic acetogenesis fractionates stronger. However, it has thus far only been studied in Acetobacterium woodii (2). There a mean fractionation factor of ε = −58.6‰ ± 0.7‰ was reported, with both branches of the acetyl-CoA pathway contributing equally. Thus, the fractionation factor for total acetate (ε_{acetate total} = −57.3‰ ± 2.3‰) was similar to that of the methyl group of acetate (ε_methyl = −58.2‰ ± 3.1‰). These data have frequently been used to interpret environmental isotopic signatures of acetate in, for example, lake sediments (14–19), rice paddies (20–23), and marine environments (24–27).

However, this reported value may not cover all acetogens, since they are metabolically, ecologically, and phylogenetically diverse (25, 28–30). We know from different methanogenic archaea, which likewise utilize the acetyl-CoA pathway to produce CH₄ from H₂ and CO₂, that the fractionation factors range from −25‰ (31) to −69‰ (32; reviewed in reference 9). Therefore, we speculated that chemolithotrophic acetate production may be different when diverse acetogenic bacteria are involved. To investigate this, we calculated the stable carbon isotope fractionation from substrate (total inorganic carbon [TIC]) utilization and product (acetate) formation of 11 different acetogenic bacteria in pure culture using a defined bicarbonate-buffered minimal medium with H₂ and CO₂ as sole the sources of carbon and energy.

A second aim was to validate the reproducibility of the fractionation factor. To our knowledge, the fractionation factor of pure cultures is usually reported for only a small number of replicates, which sometimes show minor deviations (2, 33, 34). We analyzed 21 biological replicates of Thermoanaerobacter kivui repeatedly grown in the same minimal medium under identical conditions. We hypothesized that the intraspecies difference should be smaller than the difference between the different strains.

MATERIALS AND METHODS

Growth conditions.

Acetonema longum DSM 6540, Acetobacterium carbinolicum DSM 2925, Acetobacterium woodii DSM 1030, Clostridium aceticum DSM 1496, Clostridium magnum DSM 2767, Moorella thermoacetica DSM 521, Moorella thermoacetica DSM 2995, Sporomusa ovata DSM 2662, Sporomusa sphaeroides DSM 2875, Thermoacetogenium phaeum DSM 12270, and Thermoanaerobacter kivui DSM 2030 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The cultures were routinely grown in the described minimal medium with the addition of a complex carbon source (glucose, fructose, or betain) under N₂ and CO₂ (80:20); for the chemolithotrophic experiments, the same minimal medium lacking the carbon source was used under H₂ and CO₂ (80:20). The cultures were grown at 55°C (M. thermoacetica, T. phaeum, and T. kivui) or at 30°C (all others). We focused on acetogenic strains isolated from environmental samples and therefore did not investigate isolates from a gut environment, which usually need nutrient supplements (such as yeast extract and rumen fluid), which may serve as an alternative carbon source and interfere with our isotope analysis. The medium contained (in grams per liter): NaHCO₃, 7.5; K₂HPO₄, 0.22; KH₂PO₄, 0.22; NH₄Cl, 0.31; (NH₄)₂SO₄, 0.22; NaCl, 0.45; MgSO₄·7H₂O, 0.09; cysteine, 0.5; Na₂S·9H₂O, 0.5; NH₄Fe(SO₄)₂, 10 mg; resazurin, 0.5 mg; and trace element solution, 10 ml/liter (pH 7.8). The trace element solution (in grams per liter) contained the following: nitrilotriacetic acid, 1.5; MgSO₄·7H₂O, 3; MnSO₄·H₂O, 0.5; NaCl 1; FeSO₄·7H₂O, 0.1; CoSO₄·7H₂O, 0.18; CaCl₂·2H₂O, 0.1; ZnSO₄·7H₂O, 0.18; CuSO₄·5H₂O, 0.01; KAl(SO₄)₂·12H₂O, 0.02; H₃BO₃, 0.01; Na₂MoO₄·2H₂O, 0.01; NiCl₂·6H₂O, 0.025; and NaSeO₃·5H₂O, 0.3 mg (pH 7.0). Cultures were grown in 120-ml serum bottles, which contained 50 ml of minimal medium. At the indicated times, gas samples (100 μl) were taken using a gas-tight syringe (VICI, Baton Rouge, LA) for analysis in a gas chromatograph and a gas chromatograph-combustion isotope ratio mass spectrometer (GC-C-IRMS); liquid samples (2 ml) were taken for high-pressure liquid chromatography (HPLC)-IRMS analysis.

Chemical and isotopic analyses.

CO₂ was detected after conversion to CH₄ with a methanizer (Ni catalyst at 350°C; Chrompack, Middelburg, Netherlands) in a GC with a flame ionization detector (Shimadzu, Kyoto, Japan). Isotope measurements of ¹³C and ¹²C in gas samples were performed on a GC-C-IRMS system (Thermo Fisher Scientific, Bremen, Germany). The principle operation was described by Brand (35) and Goevert and Conrad (4). The gas compounds in the 100-μl gas sample were first separated in a Hewlett-Packard 6890 GC using a Pora Plot Q column (27.5-m length, 0.32-mm internal diameter, and 10-μm film thickness; Chromopack, Frankfurt, Germany) at 30°C and He (99.996% purity; 2.6 ml min⁻¹) as the carrier gas. The sample was fed through the Finnigan Standard GC Combustion Interface III and the isotope ratio of ¹³C and ¹²C was analyzed in the IRMS (Finnigan MAT Deltaplus). The reference gas was CO₂ (99.998% purity) (Air Liquide, Düsseldorf, Germany), calibrated with the working standard methylstearate (Merck). The latter was intercalibrated at the Max Planck Institute for Biogeochemistry, Jena, Germany (courtesy of W. A. Brand) against the NBS-22 and USGS-24 standards and reported in the delta notation versus Vienna Pee Dee Belemnite.

$${\delta }^{13}\text{C}={10}^3\left({\text{R}}_{\text{sample}}/{\text{R}}_{\text{standard}}-1\right)$$ (1)

with R = ¹³C/¹²C of the sample and standard, respectively.

Isotopic measurements and quantification of acetate were performed on a HPLC system (Spectra System P1000 [Thermo Fisher Scientific, San Jose, CA]; Mistral [Spark, Emmen, Netherlands]) equipped with an ion-exclusion column (Aminex HPX-87-H; Bio-Rad, Munich, Germany) and coupled to Finnigan LC IsoLink (Thermo Fisher Scientific, Bremen, Germany) as described previously (36). Isotope ratios were detected on an IRMS (Finnigan MAT Deltaplus Advantage). The precision of the GC-IRMS was ±0.2‰, and that of the HPLC-IRMS was ±0.3‰.

Calculations.

Total inorganic carbon was distributed among the following different carbon species: gaseous CO₂ (CO_2(g)), dissolved CO₂ (CO_2(d); contains dissolved CO₂ and carbonic acid), HCO₃⁻, and CO₃²⁻. To determine the total amount of inorganic carbon (TIC; equation 5), the distribution of carbon among these species was calculated from the CO_2(g) and the pH using solubility and equilibrium constants (37); K values for 30°C were extrapolated from the tabulated values:

$${\text{CO}}_{2\left(\text{g}\right)}\leftrightarrow {\text{CO}}_{2\left(\text{d}\right)};{\text{K}}_0={10}^{-1.53}$$ (2)

$${\text{CO}}_{2\left(\text{d}\right)}+{\text{H}}_2\text{O}\leftrightarrow +{\text{H}}^{+}+{\text{HCO}}_3^{-};{\text{K}}_1={10}^{-6.36}$$ (3)

$${\text{HCO}}_3^{-}\leftrightarrow {\text{H}}^{+}+{\text{CO}}_3^{2-};{\text{K}}_2={10}^{-10.30}$$ (4)

$$\text{n}\left(\text{TIC}\right)=\text{n}\left({\text{CO}}_{2\left(\text{g}\right)}\right)+\text{n}\left({\text{CO}}_{2\left(\text{d}\right)}\right)+\text{n}\left({\text{HCO}}_3^{-}\right)+\text{n}\left({\text{CO}}_3^{2-}\right)$$ (5)

(For 55°C, K₀ = 10^−1.84, K₁ = 10^−6.24, and K₂ = 10^−10.06 were extrapolated from tabulated values [37].)

Fractionation factors for a reaction A → B are defined as described previously (38):

$${\alpha }_{A/B}=\left({\delta }_A+1,000\right)/\left({\delta }_B+1,000\right)$$ (6)

and also expressed as ε 10³ (1 − α). The isotope fractionation factor ε associated with acetogenesis was determined as described by Mariotti et al. (39) from the residual reactant:

$${\delta }_r={\delta }_{ri}+\epsilon \left[\text{In}\left(1-f\right)\right]$$ (7)

and from the product formed:

$${\delta }_p={\delta }_{ri}-\epsilon \left(1-f\right)\left[\text{In}\left(1-f\right)\right]/f$$ (8)

where δ_ri is the isotope composition of the reactant (TIC) at the beginning, and δ_r and δ_p are the isotope compositions of the residual TIC and the pooled acetate, respectively, at the instant when f was determined. Linear regression of δ_r against ln(1 − f) and of δ_p against (1− f)[ln(1 − f)]/f gives ε as the slope of best-fit lines. In a closed system the amount of used substrate (f) can be derived from the measured isotopic composition (2).

$$f=\left({\delta }_{ri}-{\delta }_r\right)/\left({\delta }_p-{\delta }_r\right)$$ (9)

Because total oxidized carbon was distributed among different carbon species (gaseous CO₂, dissolved CO₂, HCO₃⁻, and CO₃²⁻), the δ¹³C of the total inorganic carbon (δ_TIC) could not be determined directly. This value was calculated by the following mass-balance equation:

$${\delta }_{TIC}=X_g{\delta }_g+X_d{\delta }_d+X_b{\delta }_b+X_c{\delta }_c$$ (10)

where X is the mole fraction and δ is the isotopic composition of the carbon indicated by italic letters as follows: g = gaseous CO₂, d = dissolved CO₂, b = HCO₃⁻, and c = CO₃²⁻. The distribution of carbon among these species was calculated using solubility and equilibrium constants (37). δ_g was measured directly, and the remaining isotopic compositions were calculated from the relevant equilibrium isotope fractionation factors at 30°C (2, 40, 41):

$$\left({\delta }_d+1,000\right)/\left({\delta }_g+1,000\right)=0.999$$ (11)

$$\left({\delta }_b+1,000\right)/\left({\delta }_g+1,000\right)=1.0075$$ (12)

$$\left({\delta }_c+1,000\right)/\left({\delta }_g+1,000\right)=1.0061$$ (13)

(the values for 55°C are 0.994, 1.0052, and 1.0044, respectively).

On average, these corrections shifted the measured δ for CO₂ by roughly +5‰ to +7‰ for 55 and 30°C, respectively. The calculated TIC values were verified by occasionally acidifying a sample and directly determining the isotopic signal of the released CO₂. The pH of the incubations was stable over the time course (8.0 ± 0.2). In this range, a pH shift of 0.1 would change the calculated isotopic signature of TIC by ca. 0.2‰.

RESULTS

Eleven acetogenic cultures were grown in bicarbonate-buffered minimal medium with H₂ and CO₂ (80:20) as carbon and energy sources. As a typical example, Fig. 1 shows the mass flow and isotopic signature of A. woodii DSM 1030 and T. kivui DSM 2030. All other analyzed strains (except for M. thermoacetica DSM 521; see below) had a similar stoichiometry, i.e., that of the acetyl-CoA pathway:

$$4{\text{H}}_2+2{\text{CO}}_2={\text{CH}}_3\text{COOH}+2{\text{H}}_2\text{O}$$ (14)

The initial amount of 2.5 mmol of hydrogen (6.2 kPa) resulted in the production of 0.63 mmol of acetate (31.5 mM). Note that the 1.25 mmol of carbon needed for the reaction, originated both from the CO₂ of the headspace (initial, 0.6 mmol; compare Fig. 1) and the bicarbonate pool of the buffer. The TIC changed from 5.1 to 3.8 mmol, i.e., 1.3 mmol. In Fig. 1, the actual measured values of CO₂ are given as measured in the headspace. For later regression analysis, the TIC was calculated according to equation 5; the isotopic values were likewise corrected using equation 10.

Fig 1.

Measured substrate consumption (CO₂ and H₂) and product formation (acetate) (A and C) and δ¹³C values (B and D) during chemolithotrophic growth of two acetogenic strains. Values are means of three replicates and given in mmol per bottle. The CO₂ was analyzed in the headspace, and the total acetate was measured in the liquid phase. Isotopic data are given as δ in ‰.

The δ¹³C of the substrate increased with time (Fig. 1). The light carbon atoms were preferentially used to produce acetate. The isotopic signature of the produced acetate was obtained by analyzing the liquid phase. The δ¹³C of the acetate initially decreased and only at the end of the incubations increased again. This unexpected initial decrease in acetate may originate from (i) transfer of heavy acetate during inoculation, (ii) transient formate formation, or (iii) exchange of the carboxyl-group of acetate with the carbon pool (a detailed discussion can be found in the supplemental material).

To investigate the reproducibility of our analysis, we investigated 21 biological replicates of T. kivui grown on H₂ and CO₂ in the same minimal medium (seven different batches) under identical incubation conditions. Figure 2 gives the isotopic signature of all replicates plotted over the fractional yield (f_delta) of the reaction. The fractionation of the 21 T. kivui replicates ranged between ε_TIC = −62.8 and −46.6‰ (average, −53.8‰) (see Table S2 in the supplemental material) covering almost the same range as that of the different strains (see below). Although the standard deviation of the regression analysis of single cultures was generally below ±0.5‰, there were other factors influencing the fractionation among the triplicates (up to ±5‰) and, in addition, small changes in the culture media (between the seven sets) and environmental conditions influenced the reproducibility of fractionation factors (roughly ±4‰).

Fig 2.

Values of δ¹³C during the conversion of TIC to acetate in 21 replicate cultures of Thermoanaerobacter kivui. The seven batches with three replicates each are depicted in different colors (see details in Table S2 in the supplemental material). The fractional yield (f) of the reaction was calculated using equation 9. The fractionation factor was obtained by linear regression using equations 7 and 8. The data are normalized by subtracting the initial substrate signatures (δ_t0) from those of the substrate (TIC) and product (acetate) data of the individual replicates. Substrate, open diamond; product, cross. (A) Isotopic signature over fractional yield f_delta. (B and C) Mariotti plots of substrate (TIC) (B) and product (acetate) (C) data.

In Table 1 the fractionation factors of 10 different strains of acetogens are summarized. The seven mesophilic acetogens exhibited a range of fractionation factors covering roughly 15‰. Based on substrate consumption, the ε_TIC was between −68.3‰ (S. sphaeroides) and −54.0‰ (A. carbinolicum). Based on product formation, the ε_acetate ranged between −60.7‰ (S. sphaeroides) and −52.2‰ (A. carbinolicum) (Table 1). The average fractionation factor of all seven mesophilic strains was ε_TIC = −61.3‰ ± 4.3‰ and ε_acetate = −57.7‰ ± 3.1‰. The average fractionation factor determined from product formation was thus slightly lower than that of substrate utilization (due to regression analysis [see the discussion in the supplemental material on the product data]).

Table 1.

Average and standard deviation of fractionation factors (ε) for 10 different acetogens, calculated for substrate (total inorganic carbon) consumption and product (acetate) formation^a

Acetogen	εTIC		εacetate
	Avg	SD	Avg	SD
Acetobacterium carbinolium	–53.9	1.2	–52.2	4.2
Acetobacterium woodii	–60.1	1.1	–58.7	1.0
Clostridium aceticum	–58.2	1.4	–56.6	1.0
Clostridium magnum	–61.5	1.1	–59.1	1.7
Acetonema longum	–60.6	1.9	–54.9	0.7
Sporomusa ovata	–60.6	0.6	–59.9	1.1
Sporomusa sphaeroides	–68.3	3.5	–60.7	0.9
Moorella thermoacetica DSM 521	–38.2	1.6	–38.9	2.2
Moorella thermoacetica DSM 2955	–51.2	1.0	–49.1	2.5
Thermoanaerobacter kivui	–54.9	1.8	–52.2	0.5

^aTIC, total inorganic carbon. The standard deviation of the individual replicates (n = 2 or 3) is also given. Individual data for the strains are given in the Table S1 in the supplemental material.

The fractionation factors determined for the thermophiles were rather heterogeneous. Those of T. kivui (ε_TIC = −54.9‰ ± 1.8‰ and ε_acetate = −52.2‰ ± 0.5‰) and M. thermoacetica DSM 2955 (ε_TIC = −51.2‰ ± 1.0‰ and ε_acetate = −49.1‰ ± 2.5‰) were similar and fell at the lower edge of fractionation factors of the mesophiles. M. thermoacetica DMS 521 (ε_TIC = −38.2‰ ± 1.6‰ and ε_acetate = −38.9‰ ± 2.2‰) fractionated roughly 15‰ less than the two former ones. This difference is illustrated in Fig. 3 showing the isotopic signatures of all three thermophilic cultures plotted as a function of the fractional yield f_delta. It is apparent that the δ¹³C values in the cultures of T. kivui and M. thermoacetica DSM 2955 are clearly separated from those of M. thermoacetica DSM 521. For better comparison, the data were normalized by subtracting the initial substrate δ¹³C values in the individual replicates from both the substrate and the product δ¹³C data (this shifted all data points by roughly 9‰, forcing all TIC plots to start at the origin. Original data can be found in Table S1 in the supplemental material). Plotted are the data of two biological replicates for each strain. Our attempts to replicate the different behavior of M. thermoacetica DSM 521 were limited by poor growth; however, in these incubations the δ¹³C of the acetate was also always ca. 10‰ heavier than that in the control culture of T. kivui, suggesting a similar trend.

Fig 3.

Values of δ¹³C of TIC (substrate) and acetate (product) in cultures of three thermophilic acetogens (M. thermoacetica DSM 2955 = 2955, M. thermoacetica DSM 521 = 521, and T. kivui DSM 2030 = Tk). Note that M. thermoacetica DSM 521 shows a clearly distinct isotopic signature. For comparison, the data have been normalized by subtracting the initial substrate signature (δ_t0) from all substrate and product data. Substrate (total inorganic carbon [TIC]), black open symbols; product (acetate [Ac]), gray filled symbols; TIC, total inorganic carbon.

Thermoacetogenium phaeum was growing extremely poorly under the conditions applied. After more than 3 months of incubation, only 56 μmol of acetate had been produced (the theoretical value is 600 μmol; compare to Fig. 1). No other fermentation product was observed. The carbon signature of the substrate was almost unchanged over the whole incubation (δ_t0 = −8.4‰ ± 1.0‰; δ_t103 = −5.4‰ ± 1.9‰). The δ¹³C of the acetate decreased from −28.9‰ ± 0.7‰ to −60.9‰ ± 5.1‰ but did not increase again (Fig. 4) (for further discussion of the inverse fractionation, see the supplemental material). The expected fractionation factor can, however, be estimated by the difference of the isotopic signature of substrate minus product, which was at least Δ_δCO2-δac = −55.6‰. This signature is similar to the fractionation factors determined for T. kivui and M. thermoacetica DSM 2955.

Fig 4.

Values of δ¹³C of CO₂ (substrate) and acetate (product) in cultures of Thermoacetogenium phaeum DSM 12270. Note that in the time monitored the total acetate produced was 58 μmol, which is a tenth of the expected turnover. Gray squares, δ_CO2; open triangles, δ_acetate.

DISCUSSION

The unifying characteristic of the acetogenic bacteria is the implementation of the reductive acetyl-CoA pathway and the ability to use H₂ to reduce CO₂ to acetate. Most of the known acetogens (and all of the studied ones) belong to the class of clostridia. However, acetogens do not form functional clusters like methanogens or sulfate-reducing bacteria. Many acetogens group together with fermenting bacteria, which are not able to grow on H₂ and CO₂ (28, 42). Our study confirmed the already published data of A. woodii: ε_TIC = −59.0‰ ± 0.9‰ (2) versus ε_TIC = −60.1‰ ± 1.1‰ (the present study) and ε_acetate = −57.3‰ ± 2.3‰ (2) versus ε_acetate = −58.7‰ ± 1.1‰ (the present study). However, the aim of the present study was to determine whether the fractionation factor associated with the acetyl-CoA pathway is similar for all chemolithotrophic acetogens. We found that the studied acetogens exhibit a range of fractionation factors covering roughly 30‰ (ε_TIC = −68.3‰ to −38.2‰). There may be numerous factors influencing the fractionation of stable carbon isotopes in acetogenic bacteria. Some of these factors are discussed below.

Reproducibility of fractionation factor determination.

Even though the fractionation factors calculated for all independent T. kivui replicates covered a range of >16‰, our results suggest that different replicates of T. kivui (the same type culture, the same medium composition but different batches, or the same incubation conditions) exhibit almost the same variance as the individual triplicates (±4‰ between the seven replicates versus ±5‰ between the three parallels of each replicate). Our results show that the fractionation of stable carbon isotopes is quite constant (±4‰) over repeated replicates and not influenced by small changes of medium composition.

The fractionation factors of the three replicates of the other strains (ε_TIC, as well as ε_acetate) deviated by ±0.6‰ to ±4.2‰ and thereby confirmed the results seen with T. kivui. The difference between the substrate and product data was on average ±2.6‰. In general, this suggests a good reproducibility between the replicates and between substrate and product data. In addition, we tested different substrate ratios (H₂:CO₂) but did not find a major effect on the fractionation factors (M. B. Blaser, L. K. Dreisbach, and R. Conrad, unpublished data).

Acetogenic cultures show distinct carbon isotope fractionations.

The acetogenic pure cultures used in the present study are phylogenetically separate groups (Fig. 5). Therefore, we may speculate that the isotopic fractionation factors may correlate with the phylogenetic position of the organism. The fractionation factors (ε_TIC) of the distinct thermophilic cluster and the mesophilic acetogens were on averaged different by roughly 6‰ (when M. thermoacetica DSM 521 is excluded; otherwise, 12‰ [see Fig. 5]). This difference is in a similar range as the variation between closely related species: e.g., A. woodii and A. carbinolicum differ by roughly 6‰, and S. ovata and S. sphaeroides differ by almost 8‰. This suggests that the fractionation is mainly influenced by strain-specific variables.

Fig 5.

Cluster analysis of the used acetogenic cultures. The tree was calculated from ca. 1,300 nucleotide positions of 16S rRNA using the UPGMA (unweighted pair-group method with arithmetic averages) algorithm with Jukes-Cantor correction implemented in ARB (58). In the right panel, the isotopic fractionation factors of the 10 acetogenic strains are depicted. Symbols are used to indicate the mean fractionation factor (± the standard deviation; n = 2 or 3 [see Table 1; details of the calculation are shown in Table S1 in the supplemental material]) of the substrate ε_TIC (♢) and product ε_acetate (×) of the mesophilic and thermophilic strains (separated by a horizontal gray dashed line). The averages of the substrate (gray dashed line) and product (black line) are also indicated.

A similar picture is obtained when one compares the range of fractionation factors reported for closely related Methanosarcina barkeri species (DSM 800, DSM 804, and DSM 1538), which have been obtained in different laboratories using different medium compositions. When grown on H₂ and CO₂, the reported fractionation factors varied by 8‰ (6, 31, 43), whereas fractionation covers a range of 13‰ on acetate (4, 6, 33, 43), 12‰ on trimethylamine (43, 44), and 9‰ on methanol (6, 43). These differences would again point to a strain-specific variability rather than to a phylogenetic trait.

Similar differences may likewise explain why M. thermoacetica DSM 521 exhibited a rather distinct isotopic signature (Fig. 3). Both M. thermoacetica strains (DSM 521 and DSM 2955) originate from the first isolation by Fontaine et al. (described as Clostridium thermoaceticum [45]) and have been deposited by Zeikus et al. (DSM 2955; http://www.dsmz.de/catalogues/details/culture/DSM-2955.html?tx_dsmzresources_pi5[returnPid]=304) and Andreesen et al. (DSM 521; http://www.dsmz.de/catalogues/details/culture/DSM-521.html?tx_dsmzresources_pi5[returnPid]=304) later on.

A phylogeny based on functional genes rather than 16S rRNA gave a better correlation to fractionation factors of dichloromethane-degrading methylotrophic bacteria, as well as toluene-degrading microorganisms (46, 47). However, the acetyl-CoA pathway has two distinct initial CO₂-fixing enzymes (formate dehydrogenase and the carbon monoxide dehydrogenase/acetyl-CoA synthetase complex [48]), which both may contribute to the observed fractionation (Gelwicks et al. [2] reported that both branches of the acetyl-CoA pathway show a similar fractionation).

Mesophilic versus thermophilic acetogens.

The mesophilic acetogens exhibited fractionation factors that were on average 12‰ (TIC) and 10‰ (acetate) lighter than the mesophilic strains (see Fig. 5). Even though this difference is mainly affected by the low fractionation of M. thermoacetica DSM 521, the general trend to weaker fractionation was likewise observed for the other thermophilic strains. To validate that this is not a calculation artifact caused by the temperature corrections that we implemented, we used the equilibrium constants and the equilibrium fractionation factors of the mesophiles given above to recalculate the thermophilic strains. Even though the fractionation under these conditions is in general 2‰ stronger, it did not explain the difference between the two temperature groups which was up to 12‰.

If this difference solely originates in a temperature effect, a rather high effect of up to +0.43‰ per °C has to be assumed. Studies in our laboratory (49) showed only a very minor effect of the temperature on the fractionation of stable carbon isotopes when defined cultures (five methanogens and one acetogen) were measured at different temperatures (in preparation). In general, the temperature effect was below the error associated with the determination of the fractionation factor (regression analysis and intraspecies variation).

Likewise, a number of other studies found a low influence of temperature on the fractionation of stable isotopes, e.g., in fish (50), plants (51), daphnia (52), sulfate-reducing bacteria (53, 54), and methanogens (9). Therefore, it is unlikely that temperature influences the observed fractionation factors to a large extent.

It is also worth noting that M. thermoacetica contains cytochromes and conserves energy by proton-dependent ATP synthase, whereas T. kivui and A. woodii lack cytochromes but have membrane-bound corrinoids and use a sodium ion gradient to conserve energy via a coupled Na⁺ translocating ATP synthase (55, 56). Possibly, it is this difference in energy conservation that contributes to the low apparent fractionation of the two investigated M. thermoacetica strains.

Biogeochemical impact.

In natural environments, acetogenesis from H₂ and CO₂ is—in addition to fermentation—recognized as a major source of acetate during the anaerobic degradation of organic matter. The released acetate is usually further degraded to methane. The present study expands the view on isotopic fractionation of acetogenic bacteria. The formerly reported value of A. woodii could be confirmed and was similar to the average fractionation (−57.2‰) of all acetogenic strains. Therefore, our findings validate the previously used value for environmental modeling. Furthermore, we could show that the fractionation associated with the autotrophic acetyl-CoA pathway spans roughly 30‰ (ε_TIC = −68‰ to −38‰). Thereby, the range of fractionation is similar in magnitude to the ranges of fractionation by acetoclastic and hydrogenotrophic methanogens, covering −7‰ to −35‰ (9, 43) and −25‰ to −69‰ (31, 32), respectively. However, for models of carbon biogeochemistry in environments (oceans, lakes, and soil) with a low or moderate temperature, the range of fractionation factors of the mesophilic acetogens, which cover only a range of 15‰ (ε_TIC = −68‰ to −54‰), may be used. We nevertheless suggest that, instead of using one fixed value for modeling, one should preferentially use the range given above. This range would likewise cover the intraspecies variance determined for T. kivui.

When reviewing the published environmental δ¹³C values for acetate, two distinct patterns can be observed: On the one hand, low isotopic signatures of −53.6‰ ± 2.9‰ have been reported for sediments from Lake Plussee under H₂/CO₂ + BES (19). Likewise, we measured values of δ_acetate of −53.2‰ to −55.7‰ in anaerobic incubations of a shallow ditch sediment under a headspace of H₂ and CO₂ (80/20; L. K. Dreisbach, unpublished data). Similar incubations of Italian rice paddy soil resulted in an apparent fractionation of ε_{TIC, acetate} ≈ −42‰ (Blaser, unpublished). These results suggest that the microbiota in natural anoxic environments have the potential to fractionate in the same range as found for the pure cultures of acetogens. Hence, chemolithotrophic acetogenesis may potentially be active in natural anoxic environments, provided hydrogen is abundant.

On the other hand, the values of δ_acetate in lake sediments without the addition of substrate (hydrogen) have been found to range from −30‰ to −20‰, with the methyl group of the acetate being by about −10‰ relatively more depleted (δ_{acetate-methyl} = −35‰ to −40‰) (14–17, 19). An acidic fen soil with δ¹³C of total organic carbon of −26.6‰ showed δ_acetate values of −37.2‰ under H₂ and CO₂ versus −14.2‰ under argon (57). Usually such data are explained by mainly fermentative acetate production rather than hydrogen driven chemolithotrophic acetogenesis (19).

In summary, our study showed that fractionation factors for chemolithotrophic acetate production were generally large independent of the type of acetogen tested. Despite some variation according to the bacterial strain and the environmental condition, large-carbon-isotope fractionation during acetate formation in environmental samples can probably be taken as a good first indicator for ongoing chemolithotrophic acetogenesis.