Fractionation of stable carbon isotopes during microbial propionate consumption in anoxic rice paddy soils

Propionate is an important intermediate during the breakdown of organic matter in anoxic flooded paddy soils. Since there are only a few experiments on carbon isotope fractionation and the magnitude of the isotopic enrichment factors (ε) involved, we measured propionate conversion to acetate, CH₄ and CO₂ in anoxic paddy soils. Propionate consumption was measured using samples of paddy soil from Vercelli (Italy) and the International Rice Research Institute (IRRI, the Philippines) suspended in a phosphate buffer (pH 7.0) both in the absence and presence of sulfate (gypsum) and of methyl fluoride (CH₃F), an inhibitor of aceticlastic methanogenesis. Under methanogenic conditions, propionate was eventually degraded to CH₄, with acetate being a transient intermediate. Butyrate was also a minor intermediate. Methane was mainly produced by aceticlastic methanogenesis. Propionate consumption was inhibited by CH₃F. Butyrate and CH₄ were ¹³C-depleted relative to propionate, whereas acetate and CO₂ were ¹³C-enriched. The isotopic enrichment factors (ε_prop) of propionate consumption, determined by Mariotti plots, were in a range of −8 ‰ to −3.5 ‰. Under sulfidogenic conditions, acetate was also transiently accumulated, but CH₄ production was negligible. Application of CH₃F hardly affected propionate degradation and acetate accumulation. The initially produced CO₂ was ¹³C-depleted, whereas the acetate was ¹³C-enriched. The values of ε_prop were −3.5 ‰. It is concluded that the degradation of organic carbon via propionate to acetate and CO₂ involves only a little isotope fractionation. The results further indicate a major contribution of Syntrophobacter-type propionate fermentation under sulfidogenic conditions and Smithella-type propionate fermentation under methanogenic conditions. This interpretation is consistent with data regarding the microbial community composition published previously for the same soils.

1 Introduction

Propionate is a common intermediate of organic matter degradation in anoxic paddy soils. In the absence of sulfate reduction or methanogenesis, propionate may accumulate to millimolar concentrations (Conrad et al., 2014; Glissmann and Conrad, 2000; Nozoe, 1997). Under methanogenic conditions, propionate is degraded by fermentation. Several different biochemical pathways are conceivable for propionate fermentation (Textor et al., 1997). The major fermentation pathways are those by Syntrophobacter (Boone and Bryant, 1980) and Smithella (Liu et al., 1999), both members of Deltaproteobacteria. Syntrophobacter operates the methylmalonyl-CoA pathway, which results in randomization of the carbon positions of propionate (Houwen et al., 1991). This pathway can also be found in Desulfotomaculum sp. and Pelotomaculum sp. (Chen et al., 2005; DeBok et al., 2005; Imachi et al., 2002; Plugge et al., 2002) and apparently exists in many anoxic environments (Imachi et al., 2006; Krylova et al., 1997; Schink, 1985). Smithella, on the other hand, operates a dismutation pathway, which does not result in randomization (DeBok et al., 2001). This pathway has also been found in many anoxic environments (Gan et al., 2012; Lueders et al., 2004; Xia et al., 2019).

Propionate degradation by randomizing Syntrophobacter proceeds via succinate in the following way:

$$\begin{array}{}\text{(1)}& \mathrm{4}\text{ propionate }+\mathrm{8}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}\to \mathrm{4}\text{ acetate }+\mathrm{4}{\mathrm{CO}}_{\mathrm{2}}+\mathrm{12}{\mathrm{H}}_{\mathrm{2}}.\end{array}$$

Propionate degradation by non-randomizing Smithella proceeds by dismutation of propionate:

$$\begin{array}{}\text{(2)}& \mathrm{4}\text{ propionate }\to \mathrm{2}\text{ butyrate }+\mathrm{2}\text{ acetate}.\end{array}$$

Butyrate is then syntrophically converted (e.g., by Syntrophomonas; McInerney et al., 1981):

$$\begin{array}{}\text{(3)}& \mathrm{2}\text{ butyrate }+\mathrm{4}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}\to \mathrm{4}\text{ acetate }+\mathrm{4}{\mathrm{H}}_{\mathrm{2}}.\end{array}$$

The Smithella pathway, in total, is then

$$\begin{array}{}\text{(4)}& \mathrm{4}\text{ propionate }+\mathrm{4}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}\to \mathrm{6}\text{ acetate }+\mathrm{4}{\mathrm{H}}_{\mathrm{2}}.\end{array}$$

Propionate fermentation is thermodynamically endergonic under standard conditions and therefore requires syntrophic microbial partners that further convert the fermentation products. Under methanogenic conditions, the syntrophic partners are methanogenic archaea, which consume the products acetate and H₂. Under sulfidogenic conditions, sulfate-reducing bacteria replace the methanogens. Propionate can also be directly oxidized to CO₂ by propionate-degrading sulfate reducers. The overall reaction stoichiometry is the same for Syntrophobacter and Smithella:

$$\begin{array}{}\text{(5)}& {\displaystyle }\mathrm{4}\text{ propionate }+\mathrm{2}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}\to \mathrm{7}{\mathrm{CH}}_{\mathrm{4}}+\mathrm{5}{\mathrm{CO}}_{\mathrm{2}},\text{ or}\text{(6)}& {\displaystyle }\begin{array}{rl}\mathrm{4}\text{ propionate }& +\mathrm{7}\text{ sulfate }+\mathrm{11}{\mathrm{H}}^{+}\to \mathrm{7}{\mathrm{HS}}^{-}\\ & +\mathrm{12}{\mathrm{CO}}_{\mathrm{2}}+\mathrm{12}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}.\end{array}\end{array}$$

Note that the relative production of acetate and H₂ is different for Syntrophobacter and Smithella fermentation, being 1:3 and 3:2, respectively. Therefore, aceticlastic methanogenesis contributes relatively more than hydrogenotrophic methanogenesis when propionate is fermented by Smithella rather than Syntrophobacter. Under methanogenic conditions, propionate degradation in anoxic paddy soils operates close to the thermodynamic limits (Krylova and Conrad, 1998; Yao and Conrad, 2001). These restrictions are more severe for Syntrophobacter than for Smithella (Dolfing, 2013).

Using paddy soil from Italy and the Philippines, Liu and their coworkers (Liu et al., 2018a; Liu and Conrad, 2017) have recently shown that propionate consumption under sulfidogenic conditions is mainly achieved by Syntrophobacter species or other Syntrophobacteraceae, which first oxidize propionate to acetate and CO₂ and subsequently oxidize the accumulated acetate to CO₂. They also showed that Smithella was probably involved in methanogenic propionate degradation. The involvement of Smithella has also been shown for other paddy soils and sediments (Gan et al., 2012; Lueders et al., 2004; Xia et al., 2019). Since we used in the present study the same soils as Liu and their coworkers (Liu et al., 2018a; Liu and Conrad, 2017), we assumed that propionate degradation was achieved by the same microorganisms.

Knowledge of carbon isotope fractionation is important for the assessment of the pathways involved in anaerobic degradation of organic matter (Conrad, 2005; Elsner et al., 2005). The δ¹³C values of organic carbon, acetate and propionate in various soils and sediments were found to be similar (Conrad et al., 2014). The similarity indicates that the enrichment factors (ε) of the processes involved in both the production and consumption of propionate are probably small. The direct determination of ε values in microbial cultures of one propionate-producing bacterium and one propionate-consuming bacterium also showed low values (Botsch and Conrad, 2011). However, direct determination of ε values in environmental samples is missing. Therefore, we decided to measure isotope fractionation in methanogenic and sulfidogenic paddy soil amended with propionate along with the recording of the production of acetate, CH₄ and CO₂. We also used the treatment with methyl fluoride (CH₃F) to inhibit the consumption of acetate by methanogenic archaea (Janssen and Frenzel, 1997). Recently, we determined the microbial communities in methanogenic and sulfidogenic rice field soils, which were used for the assessment of ¹³C isotope fractionation during acetate consumption (Conrad et al., 2021). Here, we present analogous data from the same soil suspensions prepared for the propionate degradation experiments.

2 Materials and methods

2.1 Paddy soils and incubation conditions

The soil samples were from the research stations in Vercelli, Italy, and the International Rice research Institute (IRRI) in the Philippines. The sampling and soil characteristics have been described before (Liu et al., 2018b). The main soil characteristics will be given. The Italian soil is a sandy loam with a pH of 5.75, total C of 1.1 % and total N of 0.08 %. The Philippine soil is a silt loam with a pH of 6.3, total C of 1.9 % and total N of 0.2 %.

The experimental setup was exactly the same as during a previous study on acetate consumption (Conrad et al., 2021). Paddy soil was mixed with autoclaved anoxic H₂O at a ratio of 1:1 and incubated under N₂ at 25 ^∘C for 4 weeks. In a second incubation, paddy soil was mixed with autoclaved anoxic H₂O (prepared under N₂) at a ratio of 1:1, was amended with 0.07 g CaSO₄ ⋅ 2H₂O and was then incubated under N₂ at 25 ^∘C for 4 weeks. These two preincubated soil slurries were sampled and stored at −20 ^∘C for later molecular analysis (see data in Conrad et al., 2021). The preincubated soil slurries were also used (in three replicates) for the following incubation experiments. Two different sets of incubations were prepared. In the first set (resulting in methanogenic conditions), 5 mL of soil slurry preincubated without sulfate was incubated at 25 ^∘C with 40 mL of 20 mM potassium phosphate buffer (pH 7.0) in a 150 mL bottle under an atmosphere of N₂. The bottles were then amended with (i) 5 mL H₂O, (ii) 5 mL H₂O + 4.5 mL CH₃F, (iii) 5 mL 50 mM sodium propionate, and (iv) 5 mL 50 mM sodium propionate + 4.5 mL CH₃F. In the second set (resulting in sulfidogenic conditions), 5 mL of soil slurry preincubated with sulfate was incubated at 25 ^∘C with 40 mL of 20 mM potassium phosphate buffer (pH 7.0) in a 150 mL bottle under an atmosphere of N₂. The amendments were the same as above but with the addition of 200 µl of a CaSO₄ suspension corresponding to a concentration of 2.5 M (giving a final concentration of 10 mM sulfate).

2.2 Chemical and isotopic analyses

Gas samples for analysis of partial pressures of CH₄ and CO₂ were taken from the headspace of the incubation bottles after vigorous manual shaking for about 30 s using a gas-tight pressure-lock syringe, which had been flushed with N₂ before each sampling. Soil slurries were sampled, centrifuged and filtered through a 0.2 µm cellulose membrane filter and were stored frozen at −20 ^∘C for later fatty acid analysis. Chemical and isotopic analyses were performed as described in detail previously (Goevert and Conrad, 2009). Methane was analyzed by gas chromatography (GC) with a flame ionization detector. Carbon dioxide was analyzed after conversion to CH₄ with a Ni catalyst. Stable isotope analyses of ${}^{\mathrm{13}}\mathrm{C}/{}^{\mathrm{12}}\mathrm{C}$ in gas samples were performed using GC-combustion isotope ratio mass spectrometry (GC-C-IRMS). Propionate, butyrate and acetate were measured using high-performance liquid chromatography (HPLC) linked via a Finnigan LC IsoLink to an IRMS. The isotopic values are reported in the delta notation (δ¹³C) relative to the Vienna PeeDee Belemnite standard with a ${}^{\mathrm{13}}\mathrm{C}/{}^{\mathrm{12}}\mathrm{C}$ ratio (R_standard) of 0.01118: δ¹³C = ${\mathrm{10}}^{\mathrm{3}}(R_{\text{sample}}/R_{\text{standard}}-\mathrm{1})$. The precision of the GC-C-IRMS was ± 0.2 ‰, and that of the HPLC-IRMS was ± 0.3 ‰.

2.3 Calculations

Millimolar concentrations of CH₄ were calculated from the mixing ratios (1 ppmv ± 10⁻⁶ bar) measured in the gas phase of the incubation bottles: 1000 ppmv CH₄ correspond to 0.09 µmol mL⁻¹ of liquid. Note that this is the total amount of CH₄ in the gas phase relative to the liquid phase.

Fractionation factors for reaction A → B are defined after Hayes (Hayes, 1993) as follows:

$$\begin{array}{}\text{(7)}& {\mathit{\alpha }}_{\mathrm{A}/\mathrm{B}}=({\mathit{\delta }}_{\mathrm{A}}+\mathrm{1000})/({\mathit{\delta }}_{\mathrm{B}}+\mathrm{1000}),\end{array}$$

also expressed as ε ≡ 1000(1−α) (in ‰). The carbon isotope enrichment factor ε_prop associated with propionate consumption was calculated from the temporal change in δ¹³C of propionate as described by Mariotti et al. (Mariotti et al., 1981) from the residual reactant

$$\begin{array}{}\text{(8)}& {\mathit{\delta }}_{\mathrm{r}}={\mathit{\delta }}_{\text{ri}}+\mathit{\epsilon }\left[\text{ln}\right(\mathrm{1}-f\left)\right],\end{array}$$

where δ_ri is the isotopic composition of the reactant (propionate) at the beginning, and δ_r is the isotopic composition of the residual propionate; both of these are indicated at the instant when f is determined. f_prop is the fractional yield of the products based on the consumption of propionate ($\mathrm{0}<f_{\text{prop}}<\mathrm{1}$). Linear regression of δ¹³C of propionate against ln(1−f) yields ε_prop as the slope of the best-fit lines. The regressions of δ¹³C of propionate were done for data in the range of f_prop<0.7. The linear regressions were done individually for each experimental replicate (n=3) and were only accepted if r²>0.9. The ε values resulting from the replicate experiments were then averaged (± SE).

The fraction ($f_{{\mathrm{H}}_{\mathrm{2}}}$) of CH₄ derived from hydrogenotrophic methanogenesis was determined as described before (Conrad et al., 2010) using

$$\begin{array}{}\text{(9)}& \begin{array}{rl}{f}_{{\mathrm{H}}_{\mathrm{2}}}=& ({\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}}-{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}-\mathrm{ma}})/\\ & ({\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}-\mathrm{mc}}-{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}-\mathrm{ma}}),\end{array}\end{array}$$

where ${\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}}$ = δ¹³C of the total CH₄ produced; ${\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}-\mathrm{mc}}$ = δ¹³C of CH₄ produced from hydrogenotrophic methanogenesis, which is equivalent to the CH₄ produced in the presence of CH₃F; and ${\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}-\mathrm{ma}}$ = δ¹³C of CH₄ produced from aceticlastic methanogenesis. The ${\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{{\mathrm{CH}}_{\mathrm{4}}-\mathrm{ma}}$ was approximated from the δ¹³C of acetate in the presence of CH₃F assuming that the methyl group of acetate was depleted in ¹³C by 8 ‰ (Conrad et al., 2014) and that the enrichment factor (${\mathit{\epsilon }}_{{\mathrm{CH}}_{\mathrm{4}},\mathrm{ac}-\mathrm{methyl}}$) for CH₄ being produced from acetate–methyl was between 0 and −20 ‰.

Figure 1 Propionate conversion to acetate, butyrate, CH₄ and CO₂ in suspensions of paddy soil from Vercelli (Italy) after addition of propionate without sulfate (blue squares) or of propionate plus sulfate (gypsum) (red triangles) without CH₃F (open symbols) or with CH₃F (closed symbols). Controls with the addition of water only (blue or red circles) are only shown occasionally. The panels show the temporal change in (a) concentrations of propionate, (b) concentrations of acetate and butyrate (blue diamonds), (c) mixing ratios of CH₄ (1 ppmv = 10⁻⁶ bar), (d) mixing ratios of CO₂, (e) δ¹³C of propionate, (f) δ¹³C of acetate and butyrate, (g) δ¹³C of CH₄, and (h) δ¹³C of CO₂. Shown are the means ± SE (standard error).

Figure 2 Balance of (a, c) produced CH₄ and (b, d) produced acetate against the consumed propionate under (a, b) methanogenic and (c, d) sulfidogenic conditions in paddy soil from Vercelli (Italy). The open and closed symbols denote conditions in the absence and the presence of CH₃F, respectively. The black and red lines in (a) indicate aceticlastic methanogenesis after generation of acetate by either Smithella (Eq. 4) or Syntrophobacter (Eq. 1). The black and red lines in (b) and (d) indicate transient acetate production by Smithella and Syntrophobacter, respectively. The different symbols indicate three different replicates.

3 Results

3.1 Conversion of propionate under methanogenic and sulfidogenic conditions

Incubation of buffered suspensions of rice field soil from Vercelli (Fig. 1) and the IRRI (Fig. S1 in the Supplement) resulted in similar patterns of propionate degradation to acetate, CH₄ and CO₂. Under methanogenic conditions in the absence of sulfate, propionate degradation started after a lag phase of about 20 d (Fig. 1a), resulting in the production of acetate (Fig. 1b), CH₄ (Fig. 1c) and CO₂ (Fig. 1d). The formation of acetate, CH₄ and CO₂ in the absence of propionate was only very small. The accumulation of acetate was only transient, except when aceticlastic methanogenesis was inhibited by CH₃F (Fig. 1b). Similar observations were made in IRRI soil (Fig. S1a–d). The production of CH₄ was roughly equimolar to the consumption of propionate but was nearly zero when aceticlastic methanogenesis was inhibited by CH₃F (Fig. 2a). Under these conditions, acetate accumulated to nearly equimolar amounts with the consumed propionate (Fig. 2b), but in IRRI soil, acetate accumulation was less than equimolar (Fig. S2b in the Supplement). Butyrate was also a transient intermediate of propionate degradation and was produced and consumed simultaneously with acetate (Figs. 1b and S1b). However, the accumulated concentrations were small (< 0.1 mM).

In the presence of sulfate, propionate degradation started after a lag phase of only about 10 d (Fig. 1a), resulting in the accumulation of acetate (Fig. 1b) and the production of CO₂ (Fig. 1d), but CH₄ production was close to zero (Fig. 1c). Similar results were obtained with IRRI soil (Fig. S1a–d). The accumulated acetate was equimolar (slightly less than equimolar in the IRRI soil; Fig. S2d) to the consumption of propionate (Fig. 2d), but CH₄ was not accumulated (Fig. 2c). The addition of CH₃F had no effect. Butyrate was not detected. The accumulated acetate was subsequently degraded, resulting in further production of CO₂ (Fig. 1b and d).

Figure 3 Change in δ¹³C of propionate, acetate, butyrate, CO₂ and CH₄ relative to the fraction of propionate consumed (f_prop) under (a, b) methanogenic and (c, d) sulfidogenic conditions in paddy soil from (a, c) Vercelli (Italy) and (b, d) the IRRI (the Philippines). The different symbols indicate three different replicates.

3.2 Isotope fractionation during propionate degradation

After the onset of propionate degradation, the δ¹³C of propionate (Fig. 1e) and acetate (Fig. 1f) increased, indicating that the light isotope was preferentially consumed. The δ¹³C values of CO₂ also increased (Fig. 1h). The same was the case for butyrate (Fig. 1f). Similar results were obtained with IRRI soil (Fig. S1e–h). When aceticlastic methanogenesis was inhibited by CH₃F, the δ¹³C values of these compounds increased only slightly or decreased (Fig. 1e, f and h). However, the δ¹³C of CH₄ was much more negative (30–50 ‰) in the presence than in the absence of CH₃F (Fig. 1g). The δ¹³C values of CH₄ in unamended soil (H₂O control) were similar to those in propionate amended soil (Fig. 1g). To visualize the change in the ¹³C content of the metabolic products relative to the substrates, the δ¹³C values were plotted against the increasing fractions (f_prop) of propionate consumed in soil from both Vercelli (Fig. 3a) and the IRRI (Fig. 3b). The patterns of δ¹³C values against the f_prop indicated kinetic isotope fractionation. Note that the δ¹³C values of acetate and CO₂ were higher than those of propionate, whereas the values of butyrate and CH₄ were lower (Fig. 3a and b). The δ¹³C of CH₄ decreased until about 40 % of the propionate had been consumed and then increased again to its initial (low) values (−50 ‰ to −45 ‰) (Fig. 3a and b).

Under sulfidogenic conditions, only very little CH₄ was produced. Similarly to that under methanogenic conditions, the δ¹³C of propionate (Fig. 1e) and of acetate (Fig. 1f) increased after the onset of propionate degradation, indicating that the light isotope was preferentially consumed. However, the δ¹³C values of CO₂ decreased during the first 10–15 d when acetate was accumulated (Figs. 1h and S1h). Inhibition of aceticlastic methanogenesis by CH₃F had no effect on the δ¹³C of propionate and CO₂, but the values of acetate increased less than in the absence of CH₃F (Fig. 1f). Also, δ¹³C of CH₄ was lower in the presence than in the absence of CH₃F (Fig. 1g), but the amounts of CH₄ produced were only very small (Fig. 1c). The values of δ¹³C of propionate and acetate increased with increasing f_prop (Fig. 3c and d). The δ¹³C of acetate was generally higher by about 5–10 ‰ than the δ¹³C of propionate but also increased with f_prop, indicating kinetic isotope fractionation. However, the δ¹³C of CO₂ did not increase; instead, it decreased after the onset of propionate degradation, reaching about −35 ‰ when 50 % of the propionate had been consumed and when acetate accumulation had reached a maximum (Fig. 3c and d). Thereafter, δ¹³C of CO₂ increased or became constant.

Figure 4 Mariotti plots of propionate consumption under methanogenic and sulfidogenic (± CH₃F) conditions in paddy soil from (a) Vercelli and (b) the IRRI. The different symbols indicate three different replicates; the lines give the results of linear regression averaged over the replicates.

Mariotti plots of the ¹³C of propionate as a function of f_prop could be created for methanogenic and sulfidogenic incubation conditions, the latter being the case both in the absence and the presence of CH₃F (Fig. 4). The lines were straight even when more than 70 % of the propionate was consumed. Nevertheless, enrichment factors (ε) were determined only for f_prop < 0.7 and for regressions giving r² > 0.9. The ε_prop values were determined for each individual incubation and then averaged over the replicates (n = 2–3). The results for Vercelli and IRRI soils are summarized in Fig. 5. The average ε_prop values under methanogenic conditions were about −8 ‰ for Vercelli and about −3.5 ‰ for IRRI soil. The average ε_prop values under sulfidogenic conditions were around −3.5 ‰ in both soils, irrespective of whether CH₃F was present or not.

3.3 Hydrogenotrophic methanogenesis

The difference between the δ¹³C of CH₄ in the presence and in the absence of CH₃F was used together with the δ¹³C of acetate to roughly estimate the percentage of CH₄ derived from ${\mathrm{H}}_{\mathrm{2}}/{\mathrm{CO}}_{\mathrm{2}}$ versus acetate (Fig. S3 in the Supplement). The percentage fractions of hydrogenotrophic methanogenesis ($f_{{\mathrm{H}}_{\mathrm{2}}}$) in Vercelli soil reached a maximum after 40–50 d when acetate concentrations also reached a maximum (Fig. S3a) and then decreased strongly. The same was the case in IRRI soil after around 35 d (Fig. S3b). When assuming a reasonable isotopic enrichment factor of ${\mathit{\epsilon }}_{{\mathrm{CH}}_{\mathrm{4}},\mathrm{ac}-\mathrm{methyl}}$ = −15 ‰, which is in between the ${\mathit{\epsilon }}_{{\mathrm{CH}}_{\mathrm{4}},\mathrm{ac}-\mathrm{methyl}}$ of aceticlastic Methanosaeta (Penning et al., 2006; Valentine et al., 2004) and Methanosarcina species (Gelwicks et al., 1994; Goevert and Conrad, 2009), the average $f_{{\mathrm{H}}_{\mathrm{2}}}$ values were 0 % for Vercelli soil and 20 % for IRRI soil (Fig. S3c).

Figure 5 Isotopic enrichment factors (ε_prop, given as negative values) in paddy soils without and with the addition of sulfate (gypsum) and CH₃F. Shown are the means ± SE (standard error). The differences between the incubations were examined using Tukey's post hoc test of a one-way analysis of variance (ANOVA). Different letters on top of the bars indicate significant difference (P < 0.05) between the data.

4 Discussion

Pathway of propionate degradation

Our results showed that propionate was degraded via acetate as the main transient intermediate, finally resulting in the production of CH₄ and CO₂ under methanogenic conditions and CO₂ under sulfidogenic conditions. These results are consistent with previous observations by Liu and Conrad (Liu and Conrad, 2017) using the same paddy soils. Stable isotope probing and correlation network analysis of the microbial communities have shown that propionate degradation is accomplished by both Syntrophopbacter and Smithella species (Gan et al., 2012; Liu and Conrad, 2017; Lueders et al., 2004). The present study showed that propionate degradation under methanogenic conditions was consistent with the major operation of the Smithella pathway. The main argument for this conclusion is the observation that butyrate was a transient intermediate of propionate degradation, albeit at low concentrations (Figs. 1 and S1). In the Smithella pathway, butyrate is further fermented to acetate and H₂. However, production of H₂ is smaller in the Smithella pathway than in the Syntrophobacter pathway, while production of acetate is larger. Indeed, aceticlastic methanogenesis explained all the propionate-driven methanogenesis in the paddy soils (Figs. 2a and S2a). The average hydrogenotrophic methanogenesis, by contrast, contributed almost zero in Vercelli soil and only about 20 % in IRRI soil (Fig. S3c). The relatively larger contribution of aceticlastic compared to hydrogenotrophic methanogenesis to methanogenic propionate degradation supports the conclusion that the Smithella pathway was dominant over the Syntrophobacter pathway. Arguments against the Smithella pathway are that the accumulated CH₄ amounted to less than the expected 1.75 mole CH₄ per mole propionate consumed in Vercelli soil (Fig. 2a) and to even less in IRRI soil (Fig. S2a). With the inhibition of aceticlastic methanogenesis, acetate accumulation in Vercelli soil accounted for about 1 mole acetate per mole propionate, which is in a range that is compatible with propionate fermentation by either Smithella or Syntrophobacter (Fig. 2b). In IRRI soil, however, acetate accumulation accounted for less than 1 mole acetate per mole propionate (Fig. S2b). Note, however, that the accumulation of acetate reflects only that part of propionate fermentation, which was not inhibited by CH₃F. Our conclusion that propionate was degraded mainly by Smithella under methanogenic conditions is consistent with the microbial community structures in the paddy soils from Vercelli and the IRRI, which contain not only Syntrophobacter species but also Smithella together with Syntrophomonas, which is able to ferment butyrate (Liu and Conrad, 2017).

Under sulfidogenic conditions, propionate can be oxidized in different ways, either directly by sulfate reducers forming acetate and CO₂ or syntrophically, as under methanogenic conditions, but with subsequent oxidation of H₂ and acetate by sulfate reducers. Using the same paddy soils, Liu and their coworkers (Liu et al., 2018a; Liu and Conrad, 2017) recently showed that, under sulfidogenic conditions, propionate consumption was mainly achieved by Syntrophobacter spp., which first oxidized propionate to acetate and CO₂ and subsequently oxidized the accumulated acetate to CO₂. These were exactly the processes observed in the present study, where propionate degradation initially resulted in almost equimolar accumulation of acetate (Fig. 2d) according to

$$\begin{array}{}\text{(10)}& \begin{array}{rl}\mathrm{4}\text{ propionate }& +\mathrm{3}\text{ sulfate }+\mathrm{3}{\mathrm{H}}^{+}\to \mathrm{3}{\mathrm{HS}}^{-}\\ & +\mathrm{4}\text{ acetate }+\mathrm{4}{\mathrm{CO}}_{\mathrm{2}}+\mathrm{4}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}.\end{array}\end{array}$$

It was interesting that CH₃F was not only a strong inhibitor of aceticlastic methanogenesis (which was expected) but also a relatively strong inhibitor of propionate fermentation, though this was only the case under methanogenic conditions and not under sulfidogenic conditions. Inhibition of propionate fermentation under methanogenic conditions has been observed before in three different paddy soils and has been interpreted as being due to the adverse thermodynamic conditions when acetate accumulates (Conrad et al., 2014). However, this interpretation cannot be true since the accumulation of acetate also occurred under sulfidogenic conditions, where CH₃F did not inhibit propionate degradation. In fact, it is mainly the accumulation of H₂ rather than acetate to which propionate degradation is thermodynamically sensitive. This is the reason why the Smithella pathway is less sensitive to thermodynamic inhibition than the Syntrophobacter pathway (Dolfing, 2013). However, CH₃F did not inhibit H₂ consumption by methanogens, as seen by the low δ¹³C of CH₄ in the presence of CH₃F. Furthermore, the first step of the Smithella-type propionate fermentation does not produce any H₂, and therefore, propionate in the presence of CH₃F should at least be fermented to butyrate and acetate; however, this was not the case. Hence, the reason why CH₃F inhibited propionate fermentation under methanogenic conditions but not under sulfidogenic conditions remains unknown. Perhaps it is Smithella being more sensitive to CH₃F than Syntrophobacter.

Fractionation during propionate degradation

The isotopic fractionation of propionate apparently followed Raleigh distillation, which is characteristic for kinetic isotope fractionation in a closed system. The isotopic enrichment factor, which was determined from Mariotti plots, was in the range of ε_prop = −8 ‰ to −3.5 ‰, which is less than the enrichment factor for methanogenic acetate consumption, which has been found to be ε_ac = −21 ‰ to −17 ‰ (Conrad et al., 2021). The ε_prop values are on the same order as those predicted from δ¹³C values of propionate, acetate and organic carbon measured in various methanogenic soils and sediments (Conrad et al., 2014). Propionate degradation resulted in the formation of ¹³C-enriched acetate and CO₂ and ¹³C-depleted butyrate and CH₄. The formation of ¹³C-depleted butyrate can be explained by the kinetic isotope effect with the preferential utilization of ¹³C-depleted propionate in the initial dismutation reaction by Smithella. However, the production of ¹³C-enriched acetate cannot be explained by a linear kinetic isotope effect. We assume that the dismutation of propionate is a branch point (Fry, 2003; Hayes, 2001) at which the carbon flow is split into the production of ¹³C-enriched acetate and ¹³C-depleted butyrate. At the branch point, the carbon isotope flow shows a preferential flow of ¹²C into the product generated by the reaction with the larger fractionation factor, which would be butyrate. The further conversion of butyrate should produce acetate that is depleted in ¹³C. This acetate together with the acetate produced from propionate dismutation should result in the δ¹³C–acetate that is observed. The total acetate pool initially had a δ¹³C that was up to 10 ‰ heavier than the δ¹³C of propionate. In the end, the δ¹³C values were about equal. The observation that acetate was ¹³C-enriched relative to propionate is consistent with δ¹³C data for various soils and sediments (Conrad et al., 2014) reporting that acetate is, on average, enriched by 6 ‰ relative to propionate. Acetate was further converted to CH₄ and to CO₂. In Vercelli soil, the δ¹³C of CH₄ was about 25–35 ‰ lighter than the δ¹³C of acetate. In IRRI soil, ¹³C depletion was even larger (30–40 ‰). In both soils, the isotopic enrichment factors for acetate consumption were in a range of −12 ‰ to −17 ‰ and, for CH₄ production from acetate, they were in a range of −37 ‰ to −27 ‰ (Conrad et al., 2021). Considering that a certain percentage (albeit small) of CH₄ was formed from CO₂ reduction by hydrogenotrophic methanogenesis, which displays relatively negative enrichment factors (see the δ¹³C of CH₄ in the presence of CH₃F – Fig. 1g), the observed difference in the δ¹³C of CH₄ versus acetate is reasonable. In Smithella fermentation, the only CO₂ production occurs during the fermentation of butyrate and the aceticlastic conversion of acetate. In both cases, CO₂ should be ¹³C-depleted relative to the substrates. Note that this was not the case. Unfortunately, the ¹³C contents of the individual C atoms of propionate, butyrate and acetate are not known. The ¹³C content in the different C positions might also affect the δ¹³C of CH₄ and CO₂, which are formed. It is also possible that, besides Smithella fermentation, the Syntrophobacter fermentation contributed to propionate degradation. In summary, the detailed process of isotope fractionation during the pathway of propionate degradation is unclear. However, the magnitude of the enrichment factors involved was relatively small, being on the order of < 10 ‰.

Under sulfidogenic conditions, propionate was most probably degraded by Syntrophobacter spp., first to acetate, then finally to CO₂ (Liu et al., 2018a; Liu and Conrad, 2017). The carbon isotope fractionation of propionate consumption was, with an enrichment factor of ε_prop = −3.5 ‰, comparatively small. Propionate was eventually converted to two carbon products, of which one was depleted (the CO₂) and the other was enriched (the acetate) in terms of ¹³C. In case of Syntrophobacter-type degradation, acetate and CO₂ are produced from the conversion of pyruvate, which is generated in the methylmalonyl-CoA pathway. In this pathway, CO₂ is first consumed by the conversion of propionyl-CoA to methylmalonyl-CoA and is then produced by the conversion of oxaloacetate to pyruvate. Pyruvate is finally converted to acetate and CO₂, which should both be ¹³C-depleted with respect to pyruvate (DeNiro and Epstein, 1977). However, both acetate and CO₂ were initially ¹³C-enriched relative to propionate (about 2–5 ‰) and then changed in opposite directions, with acetate becoming increasingly ¹³C-enriched and CO₂ becoming increasingly ¹³C-depleted until the time when acetate accumulation reached a maximum (Fig. 5). Then, δ¹³C of both acetate and CO₂ increased together with the increase in ¹³C of propionate (Fig. 5). The increase in δ¹³C of acetate is often explained by consumption, especially through aceticlastic methanogenesis (Heuer et al., 2010; Heuer et al., 2009). However, hardly any CH₄ was produced under sulfidogenic conditions, and the ¹³C enrichment occurred during the phase of acetate accumulation. Therefore, the enrichment likely happened during acetate production from propionate degradation. The increasing ¹³C depletion of CO₂ can also not be explained by consumption but only by the production from propionate. Hence, isotope fractionation during the conversion of propionate, particularly during the conversion of propionate to pyruvate, is unclear. We assume complications during the carboxylation and decarboxylation reactions. Unfortunately, we found hardly any literature data on the isotope fractionation of propionate fermentation. A co-culture of Syntrophobacter fumaroxidans with Methanobacterium formicicum exhibited marginal propionate fractionation with ε_prop =  0.9 ‰ and the formation of acetate that was slightly ¹³C-enriched (about 5 ‰) (Botsch and Conrad, 2011), similarly to what is observed here. In summary, the mechanism of isotope fractionation during the conversion of propionate is not completely clear, but the magnitude of isotope fractionation is quite low.

5 Conclusions

Propionate degradation under sulfidogenic conditions was explained by the metabolism of Syntrophobacteraceae, which, in a first step, converted propionate to ¹³C-enriched acetate and ¹³C-depleted CO₂. By contrast, propionate degradation under methanogenic conditions was at least partially due to metabolism by Smithella, which, in a first step, converted propionate to ¹³C-enriched acetate and ¹³C-depleted butyrate. However, the isotopic enrichment factors (ε_prop) of propionate consumption in two paddy soils were generally very low (−8 ‰ to −3.5 ‰) under both methanogenic and sulfidogenic conditions. This low range is consistent with literature values of δ¹³C collected for propionate, acetate and organic carbon in various soils and sediments (Conrad et al., 2014). Fractionation of propionate carbon actually seems to be smaller than fractionation of acetate, which is at least 2 times larger (Conrad et al., 2021). Hence, degradation of organic carbon via propionate to acetate and CO₂ apparently involves only a little isotope fractionation on the order of < 10 ‰. By contrast, further degradation of acetate and CO₂ (+ H₂) to CH₄ involves substantial isotope fractionation. This is also the case for chemolithotrophic acetate production (Conrad et al., 2014).