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

Conrad, Ralf; Claus, Peter

doi:https://doi.org/10.5194/egusphere-2023-244

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https://doi.org/10.5194/egusphere-2023-244

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14 Feb 2023

| 14 Feb 2023

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

Ralf Conrad and Peter Claus

Abstract. Propionate is an important intermediate during the breakdown of organic matter in anoxic flooded paddy soils. Since there are only 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 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. Whereas butyrate and CH₄ were ¹³C-depleted relative to propionate, 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 degradation of organic carbon via propionate to acetate and CO₂ involves only 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 of the microbial community composition published previously for the same soils.

Received: 13 Feb 2023 – Discussion started: 14 Feb 2023

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Journal article(s) based on this preprint

31 Aug 2023

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

Ralf Conrad and Peter Claus

Biogeosciences, 20, 3625–3635, https://doi.org/10.5194/bg-20-3625-2023,https://doi.org/10.5194/bg-20-3625-2023, 2023

Short summary

Ralf Conrad and Peter Claus

Interactive discussion

Status: closed

CC1:
'Comment on egusphere-2023-244', Bernhard Schink, 07 Mar 2023

The submitted manuscript reports on isotope fractionation during propionate degradation by paddy soil samples. The results indicate contributions by Syntrophobacter-like and Smithella-like bacteria. These results complement earlier studies on microbial community analyses on the same sampling material.
The results are basically convincing although not really surprizing, given the earlier publications by the same research group on community analyses. I have only few minor corrections:
l. 115: Millimolar
l. 173: "aceticlastic methanogenesis by Smithella and Syntrophobacter" . Neither one organisms produces methane. Please, be more precise!
l. 212: and
l. 249: that the
l. 255/56: I thought that CH3F inhibits acetate conversion to CH4 and CO2!
l. 278: ...propionate in the presence of CH3F should....

Citation: https://doi.org/10.5194/egusphere-2023-244-CC1
- AC1:
  'Reply on CC1', Ralf Conrad, 22 May 2023
  
  We thank B.Schink for his helpful comments, and reply as follows.
  We intended to check the magnitude of stable isotope fractionation during anaerobic microbial propionate consumption. The overall fractionation factors were very low, and thus not very exciting. However, we think it is important to have the fractionation factors checked. In addition, patterns of isotope fractionation were different for methanogenic and sufidogenic conditions involving either Smithella or Syntrophobacter-type metabolism. We think this is an interesting observation.
  l.115: change to: ‘Millimolar’
  l.173: We agree that the phrasing is sloppy. Both microbes produce acetate and H2 in different stoichiometries (see equ. 1-4), which are subsequently converted to CH4 by methanogens. We will replace the text: aceticlastic methanogenesis after generation of acetate by either Smithella (equ.4) or Syntrophobacter (equ.1).
  l.212: change to ‘and’
  l.249: change to ‘that the’
  l.255/56: we were also surprised by the result. However, methanogenic propionate degradation was apparently inhibited by CH3F (see Fig. 1a, e) and discussion in l. 268ff.
  l.278: change to ‘…propionate in the presence of CH3F should…’
  
  Citation: https://doi.org/10.5194/egusphere-2023-244-AC1
  - RC1: 'Reply on AC1', Anonymous Referee #1, 22 May 2023
    
    ok now.
    
    Citation: https://doi.org/10.5194/egusphere-2023-244-RC1
    
    AC3: 'Reply on RC1', Ralf Conrad, 14 Jul 2023
    
    Great; Thank you!
    
    Citation: https://doi.org/10.5194/egusphere-2023-244-AC3
RC2:
'Comment on egusphere-2023-244', Anonymous Referee #2, 06 Jul 2023
This paper studied the degradation of propionate to acetate, methane and carbon dioxide in anoxic paddy soils, and conducted propionate consumption under methanogenic and sulfidogenic conditions. Carbon isotope fractionation was measured together with the above compounds. In general, the results and conclusions are sounds. I only have few comments on the methods and the formats of the manuscript to be considered before it can be published.
Line (L) 88, the soil characteristics should be summarized and briefly introduced in here, such as soil pH, total carbon, total nitrogen, etc.

L90, how did you make the anoxic H₂O?

ml should be mL; CH4 should be CH₄ in the Figure 2, and other similar problems should also be corrected.

L104-112, when and how did you get the gas samples? The detailed information should be clarified.

The legend of Figure 1 should be clearly written, some of them are repeated. Although the symbols were explained in caption, they still need to be clarified in the legend.

Figure 2 is a bit a small and hard to read.

Figure 5, statistical analyses can be done for the different treatments.
Citation: https://doi.org/10.5194/egusphere-2023-244-RC2
- AC2: 'Reply on RC2', Ralf Conrad, 10 Jul 2023
  
  We thank the anonymous reviewer for helpful comments, and reply as follows:
  The manuscript presents data from soil suspensions that were prepared exactly as for a previously published experiment on the isotope fractionation during acetate consumption. This is stated in the last sentences of the Introduction. Therefore, we described the methods only briefly by referring to this previous publication. However, there is no problem repeating some more details.
  L.88: 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%.
  L.90: Anoxic water was prepared under N2
  Typographical errors (mL instead of ml; CH4 with subscript) will be eliminated
  L.104-112: 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 stored frozen at -20C for later fatty acid analysis.
  The legend of Fig. 1 did explain the symbols besides showing them in captions. This was probably overlooked by the reviewer. Therefore, no change is required.
  We agree that Fig. 2 is a bit small. However, this was caused by the typesetting. We can only change the size of the axis labels if required. However, allowing more space for the typesetting would be preferable.
  Figure 5: statistical analysis will be added to Fig. 5: The differences between the incubations were examined using Hukey´s post hoc test of a one-way analysis of variance (ANOVA). Different letter son top of bars indicate significant difference (P <0.05) between the data.
  
  Citation: https://doi.org/10.5194/egusphere-2023-244-AC2

Interactive discussion

Status: closed

CC1:
'Comment on egusphere-2023-244', Bernhard Schink, 07 Mar 2023

The submitted manuscript reports on isotope fractionation during propionate degradation by paddy soil samples. The results indicate contributions by Syntrophobacter-like and Smithella-like bacteria. These results complement earlier studies on microbial community analyses on the same sampling material.
The results are basically convincing although not really surprizing, given the earlier publications by the same research group on community analyses. I have only few minor corrections:
l. 115: Millimolar
l. 173: "aceticlastic methanogenesis by Smithella and Syntrophobacter" . Neither one organisms produces methane. Please, be more precise!
l. 212: and
l. 249: that the
l. 255/56: I thought that CH3F inhibits acetate conversion to CH4 and CO2!
l. 278: ...propionate in the presence of CH3F should....

Citation: https://doi.org/10.5194/egusphere-2023-244-CC1
- AC1:
  'Reply on CC1', Ralf Conrad, 22 May 2023
  
  We thank B.Schink for his helpful comments, and reply as follows.
  We intended to check the magnitude of stable isotope fractionation during anaerobic microbial propionate consumption. The overall fractionation factors were very low, and thus not very exciting. However, we think it is important to have the fractionation factors checked. In addition, patterns of isotope fractionation were different for methanogenic and sufidogenic conditions involving either Smithella or Syntrophobacter-type metabolism. We think this is an interesting observation.
  l.115: change to: ‘Millimolar’
  l.173: We agree that the phrasing is sloppy. Both microbes produce acetate and H2 in different stoichiometries (see equ. 1-4), which are subsequently converted to CH4 by methanogens. We will replace the text: aceticlastic methanogenesis after generation of acetate by either Smithella (equ.4) or Syntrophobacter (equ.1).
  l.212: change to ‘and’
  l.249: change to ‘that the’
  l.255/56: we were also surprised by the result. However, methanogenic propionate degradation was apparently inhibited by CH3F (see Fig. 1a, e) and discussion in l. 268ff.
  l.278: change to ‘…propionate in the presence of CH3F should…’
  
  Citation: https://doi.org/10.5194/egusphere-2023-244-AC1
  - RC1: 'Reply on AC1', Anonymous Referee #1, 22 May 2023
    
    ok now.
    
    Citation: https://doi.org/10.5194/egusphere-2023-244-RC1
    
    AC3: 'Reply on RC1', Ralf Conrad, 14 Jul 2023
    
    Great; Thank you!
    
    Citation: https://doi.org/10.5194/egusphere-2023-244-AC3
RC2:
'Comment on egusphere-2023-244', Anonymous Referee #2, 06 Jul 2023
This paper studied the degradation of propionate to acetate, methane and carbon dioxide in anoxic paddy soils, and conducted propionate consumption under methanogenic and sulfidogenic conditions. Carbon isotope fractionation was measured together with the above compounds. In general, the results and conclusions are sounds. I only have few comments on the methods and the formats of the manuscript to be considered before it can be published.
Line (L) 88, the soil characteristics should be summarized and briefly introduced in here, such as soil pH, total carbon, total nitrogen, etc.

L90, how did you make the anoxic H₂O?

ml should be mL; CH4 should be CH₄ in the Figure 2, and other similar problems should also be corrected.

L104-112, when and how did you get the gas samples? The detailed information should be clarified.

The legend of Figure 1 should be clearly written, some of them are repeated. Although the symbols were explained in caption, they still need to be clarified in the legend.

Figure 2 is a bit a small and hard to read.

Figure 5, statistical analyses can be done for the different treatments.
Citation: https://doi.org/10.5194/egusphere-2023-244-RC2
- AC2: 'Reply on RC2', Ralf Conrad, 10 Jul 2023
  
  We thank the anonymous reviewer for helpful comments, and reply as follows:
  The manuscript presents data from soil suspensions that were prepared exactly as for a previously published experiment on the isotope fractionation during acetate consumption. This is stated in the last sentences of the Introduction. Therefore, we described the methods only briefly by referring to this previous publication. However, there is no problem repeating some more details.
  L.88: 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%.
  L.90: Anoxic water was prepared under N2
  Typographical errors (mL instead of ml; CH4 with subscript) will be eliminated
  L.104-112: 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 stored frozen at -20C for later fatty acid analysis.
  The legend of Fig. 1 did explain the symbols besides showing them in captions. This was probably overlooked by the reviewer. Therefore, no change is required.
  We agree that Fig. 2 is a bit small. However, this was caused by the typesetting. We can only change the size of the axis labels if required. However, allowing more space for the typesetting would be preferable.
  Figure 5: statistical analysis will be added to Fig. 5: The differences between the incubations were examined using Hukey´s post hoc test of a one-way analysis of variance (ANOVA). Different letter son top of bars indicate significant difference (P <0.05) between the data.
  
  Citation: https://doi.org/10.5194/egusphere-2023-244-AC2

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

ED: Publish subject to minor revisions (review by editor) (20 Jul 2023) by Jack Middelburg

AR by Ralf Conrad on behalf of the Authors (24 Jul 2023) Author's response Author's tracked changes Manuscript

ED: Publish as is (31 Jul 2023) by Jack Middelburg

AR by Ralf Conrad on behalf of the Authors (31 Jul 2023) Author's response Manuscript

Journal article(s) based on this preprint

31 Aug 2023

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

Ralf Conrad and Peter Claus

Biogeosciences, 20, 3625–3635, https://doi.org/10.5194/bg-20-3625-2023,https://doi.org/10.5194/bg-20-3625-2023, 2023

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Ralf Conrad and Peter Claus

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Ralf Conrad and Peter Claus

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Knowledge of carbon isotope fractionation is important for the assessment of the pathways involved in the degradation of organic matter. Propionate is an important intermediate. In the presence of sulfate, it was degraded by Syntrophobacter species via acetate to CO₂. In the absence of sulfate it was mainly consumed by Smithella and methanogenic archaeal species via butyrate and acetate to CH₄. However, stable carbon isotope fractionation during the degradation process was quite small.


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