Stable Carbon Isotope Fractionation by Methylotrophic Methanogenic ...

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Stable Carbon Isotope Fractionation by Methylotrophic Methanogenic Archaea Jörn Penger, Ralf Conrad, and Martin Blaser Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C1 compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (ⴚ83‰ to ⴚ72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.

B

iogenic methane release into the atmosphere is based on methane production by methanogenic archaea. The main substrates for methanogenesis are either acetate (acetoclastic methanogenesis) or hydrogen plus carbon dioxide (hydrogenotrophic methanogenesis). To a minor extent, C1 compounds such as methanol, trimethylamines, or dimethyl sulfide can also serve as methanogenic substrates (35). A number of studies indicate that less than 5 to 10% of total methanogenesis originates from methanol (24, 25, 29, 31, 38, 44). Therefore, most studies concerning environmental methane production just focus on the two main methanogenic pathways (aceticlastic and hydrogenotrophic). To distinguish them in environmental studies, methyl fluoride is widely used as a selective inhibitor of acetoclastic methanogenesis (23, 27). However, it is presently unclear how methylotrophic methanogens would react to methyl fluoride inhibition. If not affected by methyl fluoride, methylotrophic methanogens may contribute to the isotopic signal of methane, erroneously believed to be produced exclusively from CO2 reduction. Instead of specific inhibition, an alternative technique to differentiate between the substrates of methanogenesis is the determination of the difference between the stable carbon isotopes in the methanogenic substrates and the methane in environmental settings. It is believed that the so-called isotope fractionation factor (sometimes also called enrichment factor), ε, is a rather characteristic value of the individual pathways involved in carbon transformation (8, 16). A number of recent studies concerning the isotopic signature in methane production focused on the two main pathways. These studies showed a rather small fractionation range of ⫺35‰ to about ⫺5‰ for acetoclastic methanogenesis (21, 33, 41) and a comparatively broad range of fractionation of ⫺79‰ to about ⫺28‰ for hydrogenotrophic methanogenesis (34, 41). However, the very strong fractionation during hydrogenotrophic methanogenesis is probably caused by the restricted metabolism of methanogens in the late logarithmic or stationary growth phase (4, 41) or by the low energy status of the cells (34). Compared to aceticlastic and hydrogenotrophic methanogenesis, little is known about the carbon isotope fractionation during methylotrophic methanogenesis. A comparative study of Metha-

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nosarcina barkeri grown on different substrates found the strongest carbon isotope fractionation when cells were grown on methanol: for acetate, ε ⫽ ⫺22‰; for H2/CO2, ε ⫽ ⫺49‰; and for methanol, ε ⫽ ⫺79‰ (26). In accordance with that, a strong fractionation with ε of ⫺94‰ to ⫺81‰ was reported for a methylotrophic enrichment culture (36). The occurrence of methanol in the environment is based mainly on the turnover of methylated compounds of the plant cell wall, the degradation of pectin and lignin. While around 100 Tg year⫺1 of methanol is released into the atmosphere from leaves of plant vegetation, the potential source of methanol from pectin degradation of dead plants in soil was estimated to be 800 Tg year⫺1 (18). Therefore, methanol may be expected to be a common metabolite in soil environments. Especially in anoxic environments, where the degradation of plant litter is a concerted process of several bacterial guilds, methanol was found to be produced during the degradation of pectin (15, 37) and lignin (43). However, methanol can be rapidly consumed by many different microorganisms, with methylotrophic methanogens being only one of them. Even though the conversion of methanol may contribute only a relatively small percentage to total methane production, the rather large isotopic fractionation may nevertheless strongly affect the carbon isotopic readings of the produced methane. Since carbon isotope fractionation has so far been studied in only few methylotrophic methanogens (only Methanosarcina barkeri), involving only a few data points, we decided to investigate three methylotrophic methanogens: Methanosarcina barkeri, Methanosarcina acetivorans, and Methanolobus zinderi (an obligate methy-

Received 31 May 2012 Accepted 12 August 2012 Published ahead of print 17 August 2012 Address correspondence to Martin Blaser, [email protected]. Supplemental material for this article may be found at http://aem.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.01773-12

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Isotope Fractionation by Methylotrophic Archaea

FIG 1 Catabolism of methanol in pure cultures of M. acetivorans (A and B), M. barkeri (C and D), and M. zinderi (E and F). (A, C, and E) Methanol consumption, CH4 production, and concentration of CO2 in the headspace. (B, D, and F) Isotope values of methanol, CH4, and CO2. , methanol; , CH4; }, CO2. The concentrations are given as mmol per bottle; values are means and standard errors (n ⫽ 3 for Methanosarcina spp., n ⫽ 2 for Methanolobus zinderi).

lotroph). Our determination of the fractionation factor was independently based on substrate and product values. Likewise, it has been argued (but never tested) that the isotopic signal of methane under methyl fluoride inhibition can be assigned exclusively to the hydrogenotrophic methanogens (13). Therefore, we further investigated how methyl fluoride affects the carbon isotope fractionation of M. barkeri and M. zinderi. MATERIALS AND METHODS Cultures and growth conditions. Pure cultures of Methanosarcina acetivorans (type strain, DSM 2834), Methanosarcina barkeri (type strain, DSM 800), and Methanolobus zinderi (type strain, DSM 21339) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). All cultures were grown under N2-CO2 (80: 20) in 120-ml serum bottles (Ochs, Bovenden-Lenglern, Germany) filled with 50 ml medium and incubated without shaking at 37°C. Methanosarcina acetivorans was grown using a medium with the following composition (in g liter⫺1): NaCl, 23.4; MgSO4 · 7H2O, 9.4; Na2CO3, 5.0; yeast extract, 1.0; NH4Cl, 1.0; KCl, 0.9; CaCl2 · 2H2O, 0.14; Na2HPO4, 0.6; cysteine-HCl · H2O, 0.5; and Na2S · 9H2O, 0.045. In addition 12.5 ml methanol as the substrate and 1 ml trace element solution were added. The trace element solution contained (in g liter⫺1): nitrilotriacetic acid, 1.5; FeSO4 · 7H2O, 0.1; CoCl2 · 6H2O, 0.1; ZnSO4 · 7H2O, 0.1; CuSO4 · 5H2O, 0.0087; AlCl3 · 6H2O, 0.01; Na2MoO4 · 2H2O, 0.01; NiCl2 · 6H2O, 0.03; and Na2SeO4, 0.019. Methanosarcina barkeri was grown using a modified medium with the following composition (in g liter⫺1 unless otherwise noted): K2HPO4, 0.25; KH2PO4, 0.23; NH4Cl, 0.5; MgSO4 · 7H2O, 0.5; NaCl, 2.25; FeSO4 · 7H2O, 0.002; yeast extract, 2.0; Casitone, 2.0; NaHCO3, 0.85; cysteineHCl · H2O, 0.5; and Na2S · 9H2O, 0.045. In addition 10.0 ml methanol as the substrate, 1.0 ml trace element solution SL-10 (6), and 10 ml vitamin solution (45) were added. The medium used for cultivation of Methanolobus zinderi contained the following (in g liter⫺1): KCl, 0.33; MgCl2 · 6H2O, 4.0; MgSO4 · 7H2O, 3.5; NH4Cl, 0.25; K2HPO4, 0.14; NaCl, 18.0; Fe(NH4)2(SO4)2 · 6H2O, 0.002; Na-acetate, 1.0; yeast extract, 2.0; Trypticase, 2.0; NaHCO3, 5.0;

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cysteine-HCl · H2O, 0.5; and Na2S · 9H2O, 0.045. In addition 5 ml methanol as the substrate, 1.0 ml of the trace element solution used for M. acetivorans, and 10 ml vitamin solution (45) were added. For the experiments, the culture bottles were inoculated with 1 ml of a growing bacterial culture. Samples from the headspace were removed with a gas-tight syringe to determine the concentrations and carbon isotopic signatures of methane and carbon dioxide. The liquid phase was analyzed for the concentration and carbon isotopic signature of methanol. The pH of the culture liquid was also analyzed. Experiments were usually performed in triplicate. Methyl fluoride was added to the headspace as a percentage (vol/vol) of the bottle volume (120 ml). Chemical and isotopic analysis. The concentrations of CH4 and CO2 in gas samples were analyzed during the stable-isotope analysis of 13C/12C using a gas chromatograph combustion isotope ratio mass spectrometer (GC-C-IRMS) system (Thermo Fisher Scientific, Bremen, Germany). The principle of operation was described by Brand (5). The CH4 and CO2 in the gas samples (20 to 100 ␮l) were first separated in a Trace GC Ultra gas chromatograph using a Pora Plot Q column (27.5-m length, 0.32-mm inner diameter [i.d.], 10-␮m film thickness; Varian, Palo Alto, CA) at 30°C with helium (99.996% purity; 2.6 ml/min) as the carrier gas. After conversion of CH4 to CO2 in the GC Isolink 1030, the 13C/12C isotope ratio was analyzed in the IRMS (Delta V Advantage). The isotope reference gas was CO2 (99.998% purity; Air Liquide, Dusseldorf, 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 NBS 22 and USGS 24 and reported in the delta notation versus Vienna Pee Dee belemnite, ␦13C ⫽ 103 (Rsa/Rst ⫺ 1) (‰), with Rsa and Rst the 13C/12C ratio for the sample and standard, respectively. Isotopic analysis and quantification of methanol were performed in liquid samples (1 ␮l) using a second gas chromatograph combustion isotope ratio mass spectrometer (GC-C-IRMS) system (Thermo Electron, Bremen, Germany). A similar method was used by Conrad and Claus (10). The liquid sample was first evaporated in the injector at 240°C. The methanol was then separated in a Hewlett Packard 6890 GC using a Forte BP20 column (25-m length, 0.32-mm i.d., 0.5-␮m film thickness; SGE, Ringwood, Victoria, Australia) with the following temperature program:

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TABLE 1 Carbon recovery (based on measured initial methanol) and isotope enrichment factors for methanol and methane during methylotrophic methanogenesis by Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderia Organism

H3COH used (mmol)

CH4 produced (mmol)

Carbon recovery (%)

εCH3OH (‰)

εCH4 (‰)

M. acetivorans M. barkeri M. zinderi

17.14 ⫾ 0.5 12.57 ⫾ 0.5 5.85 ⫾ 0.2

9.94 ⫾ 1.2 7.55 ⫾ 0.3 3.93 ⫾ 0.3

77.32 ⫾ 6.7 80.07 ⫾ 1.0 89.69 ⫾ 2.6

⫺72.0 ⫾ 1.5 ⫺73.5 ⫾ 0.3 ⫺83.4 ⫾ 0.5

⫺71.6 ⫾ 1.2 ⫺76.1 ⫾ 1.1 ⫺77.9 ⫾ 1.8

a Values are means ⫾ standard deviations (values for isotopic calculation were taken from Table S1 in the supplemental material). Note that carbon recovery for M. zinderi was highest. This may be due to the acetate in the medium (1 g liter⫺1), which is not needed for energy metabolism but is needed for cell growth (14).

50°C, 10°C min⫺1 to 140°C, 20°C min⫺1 to 220°C, and 220°C for 1 min. Helium (99.996% purity; 2.6 ml/min) was used as the carrier gas. After conversion of methanol to CO2 in the Standard GC Combustion Interface III, the 13C/12C isotope ratio was analyzed in the IRMS (Finnigan MAT DeltaPlus). The isotope reference gas was CO2 calibrated as described above. Calculations. Fractionation factors for a reaction A ¡ B are defined as described by Hayes (22), ␣A/B ⫽ (␦A ⫹ 1,000)/(␦B ⫹ 1,000), also expressed as εA/B ' 103(1 ⫺ ␣A/B). The isotope enrichment factor ε was determined as described by Mariotti et al. (30) from the residual reactant, calculated as ␦r ⫽ ␦ri ⫹ ε[ln(1 ⫺ f)], and from the product formed, calculated as ␦p ⫽ ␦ri ⫺ ε(1 ⫺ f)[ln(1 ⫺ f)]/f, where ␦ri is the isotope composition of the reactant at the beginning and ␦r and ␦p are the isotope compositions of the residual methanol and the pooled CH4, respectively, at the instant when f was determined. f is the fractional yield of the products based on the consumption of methanol (0 ⬍ f ⬍ 1). An alternative way to calculate the fractional yield purely on the base of the measured ␦ values was promoted by Gelwicks et al. (20): fdelta ⫽ (␦ri ⫺ ␦r)/(␦p ⫺ ␦r). Linear regression of ␦r against ln(1 ⫺ f) and of ␦p against (1 ⫺ f)[ln(1 ⫺ f)]/f gives ε for substrate and product data as the slopes of best-fit lines.

RESULTS

Methylotrophic methanogenesis. Even though the times needed for growth and complete consumption of methanol were different in all three methylotrophic archaea (M. acetivorans, M. barkeri, and M. zinderi), methanol was finally completely consumed during production of methane and CO2. The following stoichiometry was observed in all three strains (Fig. 1; Table 1): 4CH3OH ¡ 3CH4 ⫹ CO2 ⫹ 2H2O. Figure 1 shows the CO2 measured in the headspace of the culture vessel, since it has been shown that CO2 rather than bicarbonate is the active substrate of methanogenesis (17, 40, 42). The total amount of inorganic carbon was larger than that in the headspace due to dissolved CO2 and bicarbonate in the medium (initial amounts of total inorganic carbon were as follows: M. barkeri, 1.1 mmol; M. acetivorans, 3.0 mmol; M. zinderi, 3.6 mmol). The growth was paralleled by a slight decrease of the pH in M. zinderi (from 7.0 to 6.7) and M. acetivorans (from 7.1 to 6.4). In contrast, there was a relatively large decrease of the pH in M. barkeri (from 6.9 to 5.2). Consumption of [12C]methanol was preferred, causing an enrichment of the heavier isotope 13C in the residual methanol (Fig. 1). Consequently, the initial CH4 produced from methanol was relatively depleted of 13C, but this then increased with time. The initially high 13C value of CH4 in the cultures may have resulted from the inoculation by transfer of dissolved CH4 or from methane produced from intracellularly stored carbon. Carbon dioxide first became slightly depleted of 13C but then became enriched with time, resulting in relatively heavy CO2 at the end of the reaction. However, CO2 was not used for determination of isotope fractionation due to the high bicarbonate background.

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The isotopic signatures recorded in the cultures of the three methanogenic strains are summarized in Fig. 2, where they are plotted as a function of the fractional yield fdelta (values are taken from Table S1 in the supplemental material). The data showed good agreement between the different strains. For regression analysis, data of all replicates of each strain were plotted together in Mariotti plots (Fig. 3). Fractionation factors were determined from the fractional regression of the ␦13C of both the substrate (εmethanol) and the product (εCH4). All three strains showed similar fractionation factors, ranging between ⫺83.4‰ and ⫺71.6‰ (the isotopic fractionation factors of the individual replicates can be found in Table S1 in the supplemental material). Lacking the isotopic signatures for low methanol concentrations (detection limit, ⬃2.5 mM [125␮mol]), we could not cover the whole range of substrate turnover. Assuming complete conversion of methanol to CH4 and CO2, the regression of methane was forced through ␦CH3OH at time zero (t0). The initial isotopic signatures of methane, which were apparently affected by methane carried over during inoculation, were not taken into account for the regression analysis (gray values in Table S1 in the supplemental material). Effect of methyl fluoride. M. barkeri and M. zinderi were grown on methanol in the presence of 0% to 3% methyl fluoride. For all M. barkeri incubations, the overall growth performance (see Fig. S1A in the supplemental material), maximal methane production rates (Table 2), and carbon flow and isotopic signature of substrate and product (Fig. 4A) were similar to those in the uninhibited samples. A slightly different picture was obtained for

FIG 2 Carbon isotope signatures in cultures of the three methylotrophic methanogens as a function of the fractional yield fdelta (showing values for all replicates). 䊐, M. acetivorans; Œ, M. barkeri; ⫻, M. zinderi. Black symbols, methanol; gray symbols, CH4; light gray symbols, CO2. (Values are taken from Table S1 in the supplemental material.)

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Isotope Fractionation by Methylotrophic Archaea

average stable-isotope fractionation factors during methanogenic methanol conversion ranged between ⫺73.6‰ and ⫺79.9‰ for M. barkeri and between ⫺74.2‰ and ⫺82.4‰ for M. zinderi. DISCUSSION

FIG 3 Mariotti plots of the substrate methanol (A) and the product methane (B) for all three methylotrophic methanogens. Linear regression gives the respective fractionation factors (ε) ⫾ standard deviation of regression. 䊐, M. acetivorans (εmethanol ⫽ ⫺72.0 ⫾ 1.5‰ and εCH4 ⫽ ⫺71.6 ⫾ 1.2‰); Œ, M. barkeri (εmethanol ⫽ ⫺73.5 ⫾ 0.3‰ and εCH4 ⫽ ⫺76.1 ⫾ 1.1‰); ⫻, M. zinderi (εmethanol ⫽ ⫺83.4 ⫾ 0.5‰ and εCH4 ⫽ ⫺77.9 ⫾ 1.8‰).

the incubations of M. zinderi: these incubations showed a prolonged lag phase under increasing methyl fluoride concentrations (up to 5 days; see Fig. S1B in the supplemental material) paralleled by a reduced maximal methane production rate (Table 2). However, the isotopic signature was not impaired by the presence of methyl fluoride (Fig. 4B). Most importantly, the isotopic fractionation of both strains was unaffected by the presence of methyl fluoride (Table 2; for details, see Tables S2A and B in the supplemental material). The

Methylotrophic methanogenesis. It is generally accepted that only a minor portion of the methane released from the environment originates from methylotrophic methanogenesis. However, this pathway might significantly contribute to the isotopic signature of total methane, since carbon isotopes seem to be strongly fractionated in this pathway. Krzycki et al. (26) obtained an fractionation of ε ⫽ ⫺74.8‰ to ⫺72.5‰ for Methanosarcina barkeri. Londry et al. (28) found a slightly lower value of ε ⫽ ⫺83.4‰ for Methanosarcina barkeri. Both studies were based on initial and endpoint measurements and did not monitor substrate consumption over time. However, our results using a closed system confirmed the previous results for M. barkeri by recording εmethanol ⫽ ⫺73.5‰ and εCH4 ⫽ ⫺76.1‰. In another Methanosarcina species, M. acetivorans, we recorded similar isotopic fractionation factors, i.e., εmethanol ⫽ ⫺72.0‰ and εCH4 ⫽ ⫺71.6‰, and the fractionation values for Methanolobus zinderi were εmethanol ⫽ ⫺83.4‰ and εCH4 ⫽ ⫺77.9‰, only slightly lower than those for the two Methanosarcina spp. In summary, we found that methylotrophic methanogens indeed fractionate carbon isotopes very strongly during the methanogenic conversion of methanol and have ε values covering a relatively narrow range of around ⫺83‰ to ⫺72‰. Among methanogenic archaea, utilization of methylated substrates is restricted to members of the family Methanosarcinaceae. The only exceptions are species of the genus Methanosphaera, which can use H2 to reduce methanol to CH4 (3). While both Methanosarcina species used in this study have a broad substrate range and can produce methane from many different substrates (e.g., from acetate, H2-CO2, methanol, methylamines, and methylated sulfides) (28), Methanolobus zinderi is an obligate methylotroph able to use only methylated compounds (14). This difference in substrate usage may in part be responsible for the observed differences in the fractionation factor. Comparing the fractionation factors expressed during CH4 production from the three methanogenic substrates acetate, H2/CO2, and methanol, it is obvious that they each cover a different range (Table 3). While acetoclastic methanogenesis is generally associated with the weakest fractionation (ε ⫽ ⫺35‰ to ⫺9‰) (21, 33, 41), hydrogenotrophic methanogenesis exhibits a broad range of fractionation factors (ε ⫽ ⫺79‰ to ⫺28‰) (41). However, methylotrophic methanogenesis has the strongest fractionation (ε ⫽ ⫺83‰ to ⫺72‰). These

TABLE 2 Highest CH4 production rates (maximal slope of methane concentration over time) and calculated fractionation factors for Methanosarcina barkeri and Methanolobus zinderi with and without methyl fluoridea M. barkeri

M. zinderi

Methyl fluoride (%)

Maximum CH4 production (mmol day⫺1)

εCH3OH (‰)

εCH4 (‰)

Maximum CH4 production (mmol day⫺1)

εCH3OH (‰)

εCH4 (‰)

0 1 2 3

1.89 1.83 1.92 1.68

⫺79.6 ⫾ 0.1 ⫺73.6 ⫾ 0.7 ⫺79.9 ⫾ 0.1 ⫺76.0 ⫾ 0.0

⫺76.9 ⫾ 1.7 ⫺77.3 ⫾ 1.7 ⫺75.0 ⫾ 2.5 ⫺75.4 ⫾ 0.6

2.66 2.64 2.07 1.36

⫺80.2 ⫾ 0.9 ⫺81.6 ⫾ 0.7 ⫺82.4 ⫾ 1.0 ⫺78.8 ⫾ 0.9

⫺75.4 ⫾ 1.6 ⫺75.1 ⫾ 1.9 ⫺74.2 ⫾ 1.7 ⫺76.5 ⫾ 6.0

a

ε values are means ⫾ standard deviations (values for isotopic calculation were taken from Table S2 in the supplemental material).

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FIG 4 Effects of different methyl fluoride concentrations on the carbon isotope signatures in cultures of M. barkeri (A) and M. zinderi (B) as a function of the fractional yield fdelta. 䊐, 0% methyl fluoride; , 1% methyl fluoride; Œ, 2% methyl fluoride; ⫻, 3% methyl fluoride. Black symbols, methanol; gray symbols, CH4; light gray symbols, CO2. (Values are taken from Table S2 in the supplemental material.)

differences in the fractionation factors may hence be useful to discriminate the different methanogenic pathways in environmental studies. The biochemical processes underlying the three methanogenic pathways (Fig. 5) show that the conversion of methanol to methane involves only two enzymes (methanol:coenzyme M methyltransferase and methyl coenzyme M reductase), while the cleavage of acetate depends on three and the reduction of CO2 on seven enzymes. All three pathways share the final step (methyl coenzyme M reductase). The only distinctive enzyme in methanogenic

conversion of methanol is the methanol:coenzyme M methyltransferase, which must be responsible for the very strong fractionation if the fractionation is a matter of enzyme function. However, it is more likely that the strong fractionation originates in the branching of the methanol pathway. The electrons needed to reduce methanol to methane originate from the concomitant oxidation of methanol to CO2 by reverting the hydrogenotrophic pathway. Therefore, it is possible that mostly the light 12C is converted to CH4, while relatively heavy carbon is converted to CO2 or is left as residual methanol. Indeed, the

TABLE 3 Compilation of carbon isotope fractionation factors (ε) for mesophilic methanogenic pure cultures grown on different carbon substrates

Organism

DSMZ strain no.

Substrate

Temp (°C)

Methanobacterium ivanovii Methanosarcina barkeri Methanosarcina barkeri Methanobacterium formicicum Methanosarcina barkeri Methanobacterium bryantii Methanococcus vanielii Methanosaeta concilii Methanosarcina barkeri Methanosarcina acetivorans Methanosarcina barkeri Methanosarcina barkeri Methanosarcina barkeri Methanosarcina acetivorans Methanosarcina barkeri Methanolobus zinderi Methanosarcina barkeri Methanosarcina barkeri Methanosarcina barkeri Methanosarcina barkeri Methanococcoides burtonii

2611 —b 804 1535 800 863 1224 3671 1538 2834 804 800 804 2834 800 21339 800 804 800 804 6242

H2-CO2 H2-CO2 H2-CO2 H2-CO2 H2-CO2 H2-CO2 H2-CO2 Acetate Acetate Acetate Acetate Acetate Acetate Methanol Methanol Methanol Methanol Methanol Trimethylamine Trimethylamine Trimethylamine

37 40 37 34 36 40 35 37 37 37 30 37 37 37 37 37 37 37 37 38 20

Fractionation factor (‰)a εsubstrate

εCH4

⫺14 ⫺24 ⫺35 ⫺31

⫺9 ⫺12 ⫺24 ⫺27

⫺72 ⫺74 ⫺83

⫺72 ⫺76 ⫺78

εCH4-substrate

Reference

⫺34 ⫺41 ⫺45c ⫺48 ⫺49 ⫺56 ⫺60d

2 19 28 1 26 19 4 33 20 21 21 26 28 This study This study This study 26 28 39 28 39

⫺22 ⫺35

⫺79 ⫺83 ⫺53 ⫺67 ⫺73

a

The calculation of the fractionation factor is based either on initial and endpoint measurements (εCH4-substrate) or on regression analysis of substrate and product data (εsubstrate and εCH4). b —, not given, but presumably DSM 800. c Value under substrate saturation; ⫺80‰ under substrate limitation. d Value in a glass fermentor; ⫺69‰ for a titanium fermentor.

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FIG 5 Schematic pathways of methanogenesis from acetate (A), H2/CO2 (41) (B), and methanol (C). Steps not required by each pathway are shaded gray. CH3-CoM, methyl coenzyme M. Redox reactions involving H2 as an electron donor are indicated by 2 e⫺. The different ranges for the fractionation factors of stable carbon isotopes obtained from methanogenic pure cultures are given in Table 3.

CO2 produced from methanol was not as depleted of 13C as the CH4. If we extrapolate the isotopic signature of the produced CO2, a signature as low as ␦CO2 newly formed ⫽ ⫺60‰ and an apparent fractionation of εCO2-methanol ⬇ ⫺20‰ can be obtained. Likewise, the strong fractionation (ranging from ⫺73‰ to ⫺53‰) of other methylated compounds, such as trimethylamines (Table 3), which differ in just the first enzyme needed to activate the methyl group may be explained by the disproportionation of the methyl compound to CO2 and CH4. Methyl fluoride and environmental implications. Theoretically, acetoclastic methanogenesis should account for 67% of total methanogenesis, when polysaccharides are completely degraded to CO2 and CH4 (7, 8). The residual CH4 production would be due to hydrogenotrophic methanogenesis, and the isotopic signature of the produced CH4 would suggest the relatively strong fractionation factors involved in CH4 production from H2-CO2. In many studies, a concentration of 2% methyl fluoride is used to inhibit acetoclastic methanogenesis (9, 11–13, 27). Applying this technique to various methanogenic aquatic sediments, fractionation factors for hydrogenotrophic methanogenesis were found to be in a range of ⫺85‰ to ⫺57‰ (9, 11, 13). Compared to these data, the apparent fractionation factors under methyl fluoride inhibition are lower by ⫺33 to ⫺9‰ than those for the uninhibited samples. Even the uninhibited samples fractionate in general more strongly than the fractionation factors reported for pure cultures of hydrogenotrophic methanogens (Table 3). One possible reason for this observation is that methylotrophic methanogenesis contributes to CH4 production in the presence of methyl fluoride, thus causing a stronger apparent fractionation. However, this option can only be relevant if methylotrophic methanogenesis is not inhibited by methyl fluoride and if stable carbon isotope fractionation is not affected by methyl fluoride. Our experiments proved that methyl fluoride indeed had no effect on the isotopic fractionation of CH4 production from methanol. Although a certain inhibition of methane production was found for M. zinderi, inhibition was not observed in M. barkeri. It is worth noting that for Methanolobus tylorii a growth-limiting effect of methyl fluoride has been observed for 3.4% but not for 1.7% methyl fluoride (32). It therefore is possible that the contribution of methylotrophic methanogenesis in the presence of methyl fluoride may affect the

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resulting isotopic signature of CH4. Let us assume that 33% of methanogenesis originates from H2-CO2 (7) and that methanol contributes up to 10% (10). In the presence of methyl fluoride, methylotrophic methanogenesis could contribute roughly 30% to total CH4 production. Under these conditions, the released methane would be on average 10‰ lighter when methylotrophic methanogens are active. Nevertheless, due to the large range of fractionation reported for hydrogenotrophic methanogens, the contribution of the methylotrophic pathway to the released methane would still be hard to judge in an environmental system. Future studies with methylotrophic archaea grown mixotrophically on various ratios of H2-CO2 and methanol could be used to further constrain the contribution of methanol to the isotopic signature of methane. Conclusion. Our results showed that three different species of methylotrophic methanogenic archaea exhibited similar fractionation factors for the methanogenic conversion of methanol and that these fractionation factors were much stronger than those reported for aceticlastic or hydrogenotrophic methanogenesis in pure cultures grown under optimal substrate conditions. Hence, even though the contribution of methanol to total methane production may be limited in the environment, methanol may nevertheless significantly affect the carbon isotopic signature of the produced CH4. Since our study showed that methyl fluoride did not affect the fractionation of methane produced from methanol, methylotrophic methanogenesis may affect the carbon isotopic signature of the produced CH4 even in the presence of methyl fluoride when acetoclastic methanogenesis is inhibited. The carbon isotopic signature of CH4 under these conditions thus may not only be due to hydrogenotrophic methanogenesis but may in addition be affected to a larger extent by methylotrophic methanogens than previously anticipated. ACKNOWLEDGMENTS We thank Peter Claus for excellent technical assistance. This study was financially supported by the Fonds der Chemischen Industrie, Germany.

REFERENCES 1. Balabane M, Galimov E, Hermann M, Letolle R. 1987. Hydrogen and carbon isotope fractionation during experimental production of bacterial methane. Org. Geochem. 11:115–119.

aem.asm.org 7601

Penger et al.

2. Belyaev SS, et al. 1983. Methanogenic bacteria from the Bondyuzhskoe oil-field: general characterization and analysis of stable-carbon isotopic fractionation. Appl. Environ. Microbiol. 45:691– 697. 3. Boone DR, Whiteman WB, Rouviere P. 1993. Diversity and taxonomy of methanogens, p 35– 80. In Ferry JG (ed), Methanogenesis— ecology, physiology, biochemistry and genetics. Chapman & Hall, New York, NY. 4. Botz R, Pokojski HD, Schmitt M, Thomm M. 1996. Carbon isotope fractionation during bacterial methanogenesis by CO2 reduction. Org. Geochem. 25:255–262. 5. Brand WA. 1996. High precision isotope ratio monitoring techniques in mass spectrometry. J. Mass Spectrom. 31:225–235. 6. Chin KJ, Rainey FA, Janssen PH, Conrad R. 1998. Methanogenic degradation of polysaccharides and the characterization of polysaccharolytic clostridia from anoxic rice field soil. Syst. Appl. Microbiol. 21:185–200. 7. Conrad R. 1999. Contribution of hydrogen to methane production and control of hydrogen concentrations in methanogenic soils and sediments. FEMS Microbiol. Ecol. 28:193–202. 8. Conrad R. 2005. Quantification of methanogenic pathways using stable carbon isotopic signatures: a review and a proposal. Org. Geochem. 36: 739 –752. 9. Conrad R, Chan OC, Claus P, Casper P. 2007. Characterization of methanogenic archaea and stable isotope fractionation during methane production in the profundal sediment of an oligotrophic lake (Lake Stechlin, Germany). Limnol. Oceanogr. 52:1393–1406. 10. Conrad R, Claus P. 2005. Contribution of methanol to the production of methane and its C-13-isotopic signature in anoxic rice field soil. Biogeochemistry 73:381–393. 11. Conrad R, Claus P, Casper P. 2010. Stable isotope fractionation during the methanogenic degradation of organic matter in the sediment of an acidic bog lake, Lake Grosse Fuchskuhle. Limnol. Oceanogr. 55:1932– 1942. 12. Conrad R, Klose M, Claus P, Enrich-Prast A. 2010. Methanogenic pathway, (13)C isotope fractionation, and archaeal community composition in the sediment of two clear-water lakes of Amazonia. Limnol. Oceanogr. 55:689 –702. 13. Conrad R, et al. 2011. Stable carbon isotope discrimination and microbiology of methane formation in tropical anoxic lake sediments. Biogeosciences 8:795– 814. 14. Doerfert SN, Reichlen M, Iyer P, Wang MY, Ferry JG. 2009. Methanolobus zinderi sp. nov., a methylotrophic methanogen isolated from a deep subsurface coal seam. Int. J. Syst. Evol. Microbiol. 59:1064 –1069. 15. Donnelly MI, Dagley S. 1980. Production of methanol from aromatic acids by Pseudomonas putida. J. Bacteriol. 142:916 –924. 16. Elsner M, Zwank L, Hunkeler D, Schwarzenbach RP. 2005. A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environ. Sci. Technol. 39:6896 – 6916. 17. Fuchs G, Thauer R, Ziegler H, Stichler W. 1979. Carbon isotope fractionation by Methanobacterium thermoautotrophicum. Arch. Microbiol. 120:135–139. 18. Galbally IE, Kirstine W. 2002. The production of methanol by flowering plants and the global cycle of methanol. J. Atmos. Chem. 43:195–229. 19. Games LM, Hayes JM, Gunsalus RP. 1978. Methane-producing bacteria: natural fractionations of the stable carbon isotopes. Geochim. Cosmochim. Acta 42:1295–1297. 20. Gelwicks JT, Risatti JB, Hayes JM. 1994. Carbon isotope effects associated with aceticlastic methanogenesis. Appl. Environ. Microbiol. 60:467– 472. 21. Goevert D, Conrad R. 2009. Effect of substrate concentration on carbon isotope fractionation during acetoclastic methanogenesis by Methanosarcina barkeri and M. acetivorans and in rice field soil. Appl. Environ. Microbiol. 75:2605–2612. 22. Hayes JM. 1993. Factors controlling 13C contents of sedimentary organic compounds—principles and evidence. Mar. Geol. 113:111–125. 23. Janssen PH, Frenzel P. 1997. Inhibition of methanogenesis by methyl fluoride: studies of pure and defined mixed cultures of anaerobic bacteria and archaea. Appl. Environ. Microbiol. 63:4552– 4557. 24. Jiang N, Wang YF, Dong XZ. 2010. Methanol as the primary methano-

7602

aem.asm.org

25. 26.

27. 28. 29. 30. 31. 32. 33. 34.

35. 36. 37. 38.

39. 40. 41. 42. 43.

44. 45.

genic and acetogenic precursor in the Cold Zoige Wetland at Tibetan Plateau. Microb. Ecol. 60:206 –213. King GM, Klug MJ, Lovley DR. 1983. Metabolism of acetate, methanol, and methylated amines in intertidal sediments of Lowes Cove, Maine. Appl. Environ. Microbiol. 45:1848 –1853. Krzycki JA, Kenealy WR, Deniro MJ, Zeikus JG. 1987. Stable carbon isotope fractionation by Methanosarcina barkeri during methanogenesis from acetate, methanol, or carbon dioxide-hydrogen. Appl. Environ. Microbiol. 53:2597–2599. Liu H, Wang J, Wang AJ, Chen JA. 2011. Chemical inhibitors of methanogenesis and putative applications. Appl. Microbiol. Biotechnol. 89: 1333–1340. Londry KL, Dawson KG, Grover HD, Summons RE, Bradley AS. 2008. Stable carbon isotope fractionation between substrates and products of Methanosarcina barkeri. Org. Geochem. 39:608 – 621. Lovley DR, Klug MJ. 1983. Methanogenesis from methanol and methylamines and acetogenesis from hydrogen and carbon dioxide in the sediments of a eutrophic lake. Appl. Environ. Microbiol. 45:1310 –1315. Mariotti A, et al. 1981. Experimental determination of nitrogen kinetic isotope fractionation—some principles. Illustration for the denitrification and nitrification processes. Plant Soil 62:413– 430. Nusslein B, Conrad R. 2000. Methane production in eutrophic Lake Plusssee: seasonal change, temperature effect and metabolic processes in the profundal sediment. Arch. Hydrobiol. 149:597– 623. Oremland RS, Culbertson CW. 1992. Evaluation of methyl fluoride and dimethyl ether as inhibitors of aerobic methane oxidation. Appl. Environ. Microbiol. 58:2983–2992. Penning H, Claus P, Casper P, Conrad R. 2006. Carbon isotope fractionation during acetoclastic methanogenesis by Methanosaeta concilii in culture and a lake sediment. Appl. Environ. Microbiol. 72:5648 –5652. Penning H, Plugge CM, Galand PE, Conrad R. 2005. Variation of carbon isotope fractionation in hydrogenotrophic methanogenic microbial cultures and environmental samples at different energy status. Glob. Change Biol. 11:2103–2113. Reeburgh WS. 2003. Global methane biogeochemistry, p 65– 89. In Keeling RF, Holland HD, Turekian KK (ed), Treatise on geochemistry, vol 4. Elsevier-Pergamon, Oxford, United Kingdom. Rosenfeld WD, Silverman SR. 1959. Carbon isotope fractionation in bacterial production of methane. Science 130:1658 –1659. Schink B, Zeikus JG. 1980. Microbial methanol formation: a major end product of pectin metabolism. Curr. Microbiol. 4:387–389. Singh N, Kendall MM, Liu YT, Boone DR. 2005. Isolation and characterization of methylotrophic methanogens from anoxic marine sediments in Skan Bay, Alaska: description of Methanococcoides alaskense sp. nov., and emended description of Methanosarcina baltica. Int. J. Syst. Evol. Microbiol. 55:2531–2538. Summons RE, Franzmann PD, Nichols PD. 1998. Carbon isotopic fractionation associated with methylotrophic methanogenesis. Org. Geochem. 28:465– 475. Thauer RK, Kaufer B, Fuchs G. 1975. The active species of ‘CO2’ utilized by reduced ferredoxin: CO2 oxidoreductase from Clostridium pasteurianum. Eur. J. Biochem. 55:111–117. Valentine DL, Chidthaisong A, Rice A, Reeburgh WS, Tyler SC. 2004. Carbon and hydrogen isotope fractionation by moderately thermophilic methanogens. Geochim. Cosmochim. Acta 68:1571–1590. Vorholt JA, Thauer RK. 1997. The active species of ‘CO2’ utilized by formylmethanofuran dehydrogenase from methanogenic archaea. Eur. J. Biochem. 248:919 –924. Warneke C, et al. 1999. Acetone, methanol, and other partially oxidized volatile organic emissions from dead plant matter by abiological processes: significance for atmospheric HOx chemistry. Glob. Biogeochem. Cycle 13:9 –17. Winfrey MR, Nelson DR, Klevickis SC, Zeikus JG. 1977. Association of hydrogen metabolism with methanogenesis in Lake Mendota sediments. Appl. Environ. Microbiol. 33:312–318. Wolin EA, Wolin MJ, Wolfe RS. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882–2886.

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