Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosaeta concilii in Culture and a Lake Sediment

Abstract

The isotope enrichment factors (ɛ) in Methanosaeta concilii and in a lake sediment, where acetate was consumed only by Methanosaeta spp., were clearly less negative than the ɛ usually observed for Methanosarcina spp. The fraction of methane produced from acetate in the sediment, as determined by using stable isotope signatures, was 10 to 15% lower when the appropriate ɛ of Methanosaeta spp. was used.

Methane is a major product of the degradation of organic matter in anoxic environments like rice paddies, natural wetlands, and lake sediments. Its production from H₂-CO₂ and acetate (ac) in natural systems has been quantified by stable isotope modeling (7, 17). One of the input parameters needed is the carbon isotope fractionation of acetoclastic methanogenesis. To determine the fractionation factor (α) in culture or in an environmental system, the δ¹³C of the methyl carbon of acetate (ac-methyl) must be known, since CH₄ is produced from the methyl carbon rather than the carboxyl carbon of acetate (5, 19). For Methanosarcina spp. isotope fractionation has been determined in pure culture (7, 10, 20) and has been verified in natural environments dominated by this group of acetoclastic methanogens (14). However, detailed data on isotope fractionation by Methanosaeta spp. are rare. Valentine et al. (18) reported a rather low fractionation factor (α = 1.007) for thermophilic Methanosaeta thermophila. Similarly, the fractionation factor for mesophilic Methanosaeta concilii also seems to be lower (α = 1.017) (A. Chidthaisong, S. C. Tyler, et al., unpublished results) than that for Methanosarcina spp. (α = 1.021 to 1.027) (for a review see reference 18). A difference in isotope fractionation by the two acetoclastic methanogenic groups is possible, since they differ in their biochemical activation of acetate to acetyl coenzyme A (acetyl-CoA).

To increase our knowledge of isotope fractionation by Methanosaeta spp., we grew M. concilii under defined conditions and determined the isotope enrichment factor (ɛ). We also determined isotope fractionation by acetoclastic Methanosaeta spp. in a natural environment, Lake Dagow sediment, in which only members of the Methanosaetaceae were detected (2, 8).

Cultures (n = 5) of M. concilii DSM 3671 were grown under N₂-CO₂ (80:20) at 37°C in glass bottles (250 ml; Müller Krempel, Bülach, Switzerland) without shaking using carbonate-buffered (pH 7.1) mineral medium (DSM medium 334). Sodium acetate was added to an initial concentration of approximately 70 mM. For the total molecule the added acetate (ac) had a δ¹³C_ac of −24.0‰, and for the methyl group the δ¹³C_ac-methyl was −29.4‰. After inoculation with a 20% bacterial suspension in the late exponential phase (resulting in a final volume of 125 ml), several gas (0.4-ml) and liquid (2-ml) samples were removed and used for analysis of pH, concentration, and the carbon isotope composition of acetate and the products formed.

Sediment samples from eutrophic Lake Dagow (Northern Brandenburg, Germany) were obtained on 9 November 2004 and 17 August 2005 and transported to the laboratory in Marburg as described previously (8). The sediment samples from 2004 (19 ml; depth, 0 to 10 cm) and from 2005 (10 ml; depth, 0 to 5 cm) were placed in 120-ml serum vials and 27-ml pressure tubes, respectively. The glass vessels were closed with butyl rubber stoppers and incubated under an N₂ atmosphere at 10°C without shaking. After preincubation overnight, the appropriate volume of a sodium acetate stock solution (40.6 mM) was added to obtain an initial acetate concentration of 1 mM. The acetate had a δ¹³C_ac of −32.1‰ and a δ¹³C_ac-methyl of −36.4‰. At each time examined during incubation triplicate tubes were analyzed and subsequently sacrificed. In another experiment, using sediment from 2005, CH₄ production was investigated after consumption of the acetate that was added. To do this, the headspace was exchanged with N₂, which was followed by repeated gas sampling over the next 18 days.

Chemical and isotopic analyses were performed as described by Penning and Conrad (13). A stable isotope analysis of ¹³C/¹²C in gas samples was performed using a gas chromatograph-combustion-isotope ratio mass spectrometer system (Thermoquest, Bremen, Germany) (13). Measurement of isotopes and quantification of acetate were performed with a high-performance liquid chromatography system coupled to Finnigan LC IsoLink (Thermo Electron Corporation, Bremen, Germany). Off-line pyrolysis was performed to determine δ¹³C_ac-methyl contents (1, 3).

The amount of total inorganic carbon (TIC) produced by M. concilii was calculated (13) and was expressed as the difference between the amount of TIC at any time and the initial amount of TIC. The isotope enrichment factor associated with acetoclastic methanogenesis was determined as described by Mariotti et al. (11) from the residual reactant

(1)

and from the product formed

(2)

where δ_ri is the isotope composition of the reactant (either ac or ac-methyl) at the beginning, δ_r and δ_p are the isotope compositions of the residual ac and the pooled CH₄, respectively, at the instant when f was determined, and f is fractional yield of the products based on the consumption of ac (0 < f < 1). Linear regression of δ_r against ln(1 − f) and of δ_p against (1 − f)[ln(1 − f)]/f gives ɛ as the slope. The enrichment factor was converted to the fractionation factor: ɛ = 10³ × (1 − α).

The relative contribution of acetate- and CO₂-derived CH₄ to total CH₄ was determined as follows (4):

(3)

where f_mc is the fraction of CH₄ formed from H₂-CO₂, δ_ma and δ_mc are the isotope ratios of CH₄ derived from either ac or H₂-CO₂, and δ_CH4-new is the isotopic signature calculated for the methane formed since the last measurement (13). δ_ma was calculated from δ_ac-methyl using α_ma (experimentally determined in this study and from previously published data). δ_mc was calculated using the isotope fractionation factor for hydrogenotrophic methanogenesis (α_CO2/CH4 = 1.085) determined previously for Lake Dagow sediment (2). For calculation of δ_ma, δ_ac-methyl was assumed to be equal to δ¹³C of organic matter (δ_org = −30.4 ± 0.4) (2).

During growth experiments with M. concilii acetate was consumed within 550 h to threshold concentrations and was converted to CO₂ and CH₄ (Fig. 1A). The amount of CO₂ and CH₄ accounted for approximately 73% of the initial amount of acetate. As expected for a closed-system approach, the δ¹³C values for both the substrate and the products increased due to continuous preferential consumption of the [¹²C]acetate (Fig. 1B). The initial high δ¹³C value of methane (δ¹³C_CH4) was due to transfer of dissolved CH₄ by inoculation. δ¹³C_CO2 increased with time but was not used for determination of isotope fractionation, since the high background level of the bicarbonate buffer did not allow precise quantification of the δ¹³C of the newly formed TIC. Isotope fractionation during CH₄ formation from acetate was determined from data for acetate, ac-methyl, and CH₄ using equations 1 and 2 (Fig. 2). The isotope enrichment factors determined for ac-methyl (ɛ_ac-methyl = −10.4‰ ± 1.5‰) and CH₄ (ɛ_CH4 = −9.4‰ ± 0.9‰) agreed within error, whereas the isotope enrichment factor for acetate (both carbon atoms) was more negative (ɛ_ac = −13.6‰ ± 0.6‰). The good correlation fit indicates that even for a wide range of concentrations (70 mM to the threshold concentration) the magnitude of isotope fractionation was basically unchanged. Isotope enrichment in the carboxyl carbon of ac (ac-carboxyl) was calculated from δ¹³C_ac-carboxyl (δ¹³C_ac-carboxyl = 2δ¹³C_ac − δ¹³C_ac-methyl) using equation 1, resulting in an ɛ_ac-carboxyl value of −16.0‰.

FIG. 1.

Catabolism of acetate in a pure culture of M. concilii. (A) Acetate consumption, product formation, and pH. (B) Isotope signatures of total acetate, ac-methyl, CO₂, and CH₄. ▴, ac; •, ac-methyl; □, CO₂; ○, CH₄; line with no symbols, pH. The values are means ± standard errors (n = 5).

FIG. 2.

Isotope enrichment during acetoclastic methanogenesis by M. concilii. The plots are based on equations derived by Mariotti et al. (11). (A) ▴, ac; •, ac-methyl. (B) CH₄. The values are means ± standard errors (n = 5).

Our study shows that the isotopic fractionation factor of acetate methyl to CH₄ was much less in M. concilii (ɛ = ∼−10‰) than in Methanosarcina barkeri (ɛ = ∼−20‰) (7). The agreement of isotope enrichment in ac-methyl (ɛ_ac-methyl) (Fig. 2A) and CH₄ (ɛ_CH4) (Fig. 2B) is a reasonable result, since in M. concilii the methyl group should be almost exclusively (98%) used for methane production (12). After complete consumption of acetate, the intercept of the regression line of CH₄ (δ_ri) (Fig. 2B) was −29.5‰, which is almost identical to δ¹³C_ac-methyl (−29.4‰), as expected from equation 2. The δ¹³C of the biomass of M. concilii was not determined in the experiment reported here, but previous studies showed that the biomass was slightly ¹³C enriched with respect to the initial δ¹³C_ac (ɛ_biomass = 6.0‰ ± 0.7‰) (unpublished data). We assumed that due to the relatively low level of biomass formation in anaerobic metabolism δ¹³C_CH4 was probably not significantly affected, but this might have resulted in slightly enhanced depletion of ¹³C in the catabolic product CH₄ (e.g., a lower ɛ for CH₄ than for ac and ac-methyl).

The depletion of ¹³C in the ac-carboxyl (ɛ_ac-carboxyl = −16.0‰) was greater than that in the ac-methyl, as observed previously for M. barkeri (7). The stronger fractionation in ac-carboxyl could theoretically have been caused by reversible exchange of the carbonyl carbon of acetyl-CoA with the ¹³C-enriched CO₂ in the growth medium. This exchange reaction was observed in M. barkeri (6). However, when we considered such an exchange reaction using a δ¹³C_ac-carboxyl value of −18.7‰ of the initial acetate and a δ¹³C_CO2 value of ≤−24‰ (Fig. 1B), we expected ¹³C depletion leading to weaker isotope fractionation in δ¹³C_ac-carboxyl, which was not the case. Therefore, the exchange reaction either might not be active in M. concilii or might be catalyzed to only a minor extent. As an alternative explanation we suggest that the stronger isotope fractionation in the ac-carboxyl was caused by ¹³C fractionation during conversion of acetate via acetyl phosphate to acetyl-CoA. The actual bond breaking of this reaction occurs at the carboxyl carbon (5), and therefore the carboxyl carbon might experience a stronger isotope effect.

The isotope fractionation during acetoclastic methanogenesis in Methanosaeta spp. (M. concilii in this study and M. thermophila in the study of Valentine et al. [18]) is apparently weaker than that in Methanosarcina spp., in which fractionation factors typically range from 1.021 to 1.027, equivalent to enrichment factors of −21 to −27‰ (7, 10, 20). Gelwicks et al. (7) proposed that the carbon monoxide dehydrogenase (CODH) enzyme complex catalyzes the rate-limiting step of the overall reaction and therefore is responsible for the observed fractionation. Yet this does not explain the difference in isotope fractionation caused by the two groups of acetoclastic methanogenic archaea. Although the CODHs of Methanosarcina spp. and Methanosaeta spp. differ to some extent, the enzymes catalyze the same principal reactions, which determine the magnitude of the isotope effect. Thus, a priori it is not expected that the two CODHs would exhibit very different isotope effects. We therefore speculated that the difference in the overall isotope fractionation is caused by the initial activation of acetate. In this reaction the two acetoclastic genera differ biochemically. While Methanosarcina spp. activate acetate in two steps (acetate kinase and phosphotransacetylase) at the expense of one high-energy phosphate bond, Methanosaeta spp. activate acetate using the acetyl-CoA synthetase at the expense of two high-energy phosphate bonds (9). Activation by Methanosarcina spp., which is driven only by the energy from the breakage of one high-energy bond, may have greater reversibility. This could explain why the isotope effect of the later C—C bond cleavage of acetyl-CoA is expressed more strongly.

Lake Dagow sediment (obtained in 2004) was incubated anoxically after addition of acetate (initial concentration, 1 mM). Under these conditions CH₄ was produced from H₂-CO₂, from acetate that is naturally produced in the sediment, and from the acetate added (Fig. 3A). Within 92 h, the elevated concentrations caused by addition of acetate decreased to constant low concentrations (93 ± 18 μM). Quantification of acetate and CH₄ showed that in the sediment samples 52% of the total CH₄ produced was derived from the additional acetate. Note that acetoclastic methanogenesis was the only acetate-consuming process in the sediment, which was devoid of sulfate or other inorganic electron acceptors (2). Due to preferential consumption of ¹²C for CH₄ production, ac, as well as ac-methyl, became enriched in ¹³C with time (Fig. 3B). Simultaneously, CH₄ also became isotopically enriched, but to a minor extent. After 50 h, δ¹³C again decreased due to dilution of the residual ¹³C-enriched acetate by acetate produced from anaerobic degradation of sediment organic matter. Since CH₄ was also produced from H₂-CO₂, isotope enrichment by acetoclastic methanogenesis was calculated only from the isotope data for acetate. Only data for f values that were ≥0 and ≤0.5 (ln[1 − f] ≥ −0.7) were analyzed, since acetate was still being produced in the sediment. At a higher f value the continuously produced isotopically relatively light acetate would dilute δ¹³C_ac-methyl and δ¹³C_ac (Fig. 3) and therefore bias the quantification of ɛ. The values of ɛ_ac and ɛ_ac-methyl determined were −12.9‰ ± 1.2‰ and −14.2‰ ± 2.2‰, respectively. The data point at a ln(1 − f) value of 0.69 influenced the determination of ɛ. Removal of this data point altered ɛ_ac to −20.3‰, yet ɛ_ac-methyl changed only slightly to −11.6‰. We also studied sediment obtained in 2005. Although the ɛ values determined were somewhat different (ɛ_ac = −17.2‰ ± 1.2‰ with r² = 0.928; ɛ_ac-methyl = −7.8‰ ± 1.8‰ with r² = 0.566), they clearly confirmed the tendency of ɛ to be greater than −20‰. Notably, the results for Lake Dagow sediment confirmed that the ɛ values for Methanosaeta spp. were clearly less than those for Methanosarcina spp. (7, 10, 20).

FIG. 3.

Acetate consumption in Lake Dagow sediment (obtained in 2004) amended with acetate. (A) Acetate consumption (▴) and CH₄ production (○). (B) Isotope ratios of total ac (▴), ac-methyl (•), CO₂ (□), and CH₄ (○). (C) The plots are based on equations derived by Mariotti et al. (11). The values in panels A and B are means ± standard errors (n = 3).

The difference between the two acetoclastic methanogenic genera is important for correct computation of the amount of CH₄ that is produced from acetate compared with the amount of CH₄ that is produced from CO₂ (equation 3). A lower ɛ_ac-methyl value leads to a higher value of f_mc. Thus, application of the fractionation factor of Methanosarcina spp. to an environment that is dominated by Methanosaeta spp. would result in underestimation of the relative contribution of H₂-CO₂-dependent CH₄ production. We tested the influence of ɛ_ac-methyl on this quantification using data from anoxic incubation of Lake Dagow sediment. We used sediment samples from 2005 and calculated the relative fraction of CH₄ produced from H₂-CO₂ (f_mc) (equation 3) during the incubation while CH₄ was produced at a constant rate (Fig. 4A). δ¹³C_CO2 in the carbonate-buffered system stayed fairly constant, while δ¹³C_CH4 decreased, reaching stabile values around −63‰. The sensitivity of f_mc to ɛ_ac-methyl was calculated using the enrichment factors either for Methanosarcina spp. (7), for M. concilii (Fig. 2A), or for Lake Dagow sediment (Fig. 4B). Comparison of calculated f_mc values showed that f_mc was on average larger by 0.11 units (ɛ_ac-methyl = −14.2‰) and 0.15 units (ɛ_ac-methyl = −10.4‰) using enrichment factors obtained in this study rather than using previously published values typical for acetoclastic Methanosarcina spp. (ɛ_ac-methyl = −20‰). The final f_mc values were 0.33, 0.42, and 0.46 for ɛ_ac-methyl values of −20‰, −14.2‰, and −10.4‰, respectively. The f_mc values of 0.2 to 0.3 calculated for an previous incubation experiment performed with Lake Dagow sediment (2) might have been biased by such an inappropriate fractionation factor, so that the real values probably are slightly higher (0.3 to 0.4), as suggested by Fig. 4.

FIG. 4.

Effect of ɛ_ac-methyl on quantification of the fraction of CH₄ originating from H₂-CO₂ (f_mc). (A) Production of CH₄ (⧫) and isotope ratios of the newly formed CH₄ (○) and of the CO₂ (□) during incubation of Lake Dagow sediment (obtained in 2005). The values are means ± standard errors (n = 3). (B) f_mc calculated using different enrichment factors (ɛ_ac-methyl) (an ɛ_ac-methyl of −14.2‰ is the value determined in this study for Lake Dagow sediment).

Our results show that two functionally equivalent groups of acetoclastic methanogens, whose biochemical pathways differ, exhibit different isotope fractionation and consequently affect isotope modeling. Hence, microbial community structure can affect biogeochemical fluxes and thus provides an example of the hypothesis that the composition of environmental microbial communities does affect ecosystem function (15, 16).