The energy-efficient reductive tricarboxylic acid cycle drives carbon uptake and transfer to higher trophic levels within the Kueishantao shallow-water hydrothermal system

Maak, Joely Marie; Lin, Yu-Shih; Schefuß, Enno; Aepfler, Rebecca F.; Liu, Li-Lian; Elvert, Marcus; Bühring, Solveig I.

doi:https://doi.org/10.5194/egusphere-2024-1356

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https://doi.org/10.5194/egusphere-2024-1356

Preprints

29 May 2024

| 29 May 2024

The energy-efficient reductive tricarboxylic acid cycle drives carbon uptake and transfer to higher trophic levels within the Kueishantao shallow-water hydrothermal system

Joely Marie Maak, Yu-Shih Lin, Enno Schefuß, Rebecca F. Aepfler, Li-Lian Liu, Marcus Elvert, and Solveig I. Bühring

Abstract. Chemoautotrophic Campylobacteria utilize the reductive tricarboxylic acid (rTCA) cycle for carbon uptake, a metabolic pathway that is more energy efficient and discriminates less against ¹³C than the Calvin-Benson-Bassham cycle. Similar to other hydrothermal systems worldwide, Campylobacteria dominate the microbial community of the shallow-water hydrothermal system off Kueishantao (Taiwan). Compound-specific carbon stable isotope analyses of lipid-derived fatty acids were performed to understand the importance of rTCA and the transfer of fixed carbon to higher trophic levels in the vent area. Of these, C_16:1ω7c, C_18:1ω7c, and C_18:1ω9 fatty acids were strongly enriched in ¹³C, indicating the activity of rTCA utilizing Campylobacteria. Isotopic fractionation was close to 0 ‰, likely caused by pH values as low as 2.88. Characteristic fatty acids were present not only in the vent fluids but also in adjacent sediments and water filters 20 m away from the vent orifice, even though with decreasing abundance and diluted ¹³C enrichment. Furthermore, δ¹³C analysis of fatty acids from the tissue of Xenograpsus testudinatus, a crab endemic to this particular vent system, identified the trophic transfer of chemosynthetically fixed carbon. This highlights the interrelationship between chemoautotrophic microbial activity and life opportunities of higher organisms under environmentally harsh conditions at shallow-water hydrothermal systems.

Received: 07 May 2024 – Discussion started: 29 May 2024

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15 Apr 2025

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The energy-efficient reductive tricarboxylic acid cycle drives carbon uptake and transfer to higher trophic levels within the Kueishantao shallow-water hydrothermal system

Joely M. Maak, Yu-Shih Lin, Enno Schefuß, Rebecca F. Aepfler, Li-Lian Liu, Marcus Elvert, and Solveig I. Bühring

Biogeosciences, 22, 1853–1863, https://doi.org/10.5194/bg-22-1853-2025,https://doi.org/10.5194/bg-22-1853-2025, 2025

Short summary Co-editor-in-chief

Joely Marie Maak, Yu-Shih Lin, Enno Schefuß, Rebecca F. Aepfler, Li-Lian Liu, Marcus Elvert, and Solveig I. Bühring

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2024-1356', Anonymous Referee #1, 11 Oct 2024

Maak and colleagues report the isotopic compositions of fatty acids in samples collected in the vicinity of a shallow-water hydrothermal system near Kueishantao (Taiwan). The samples comprised particulate organic carbon, sediments and tissues from an endemic crab. While most of the compounds analysed had d¹³C values in the range -23 to -30 per mil consistent with carbon assimilated through CBB pathway, some specific fatty acids namely C16:1ω7c, n-C16:0, C18:1ω7c, were anomalously enriched suggesting they originated from organisms assimilating carbon chemosynthetically through the rTCA pathway. The organisms producing these fatty acids are identified as sulfur oxidising Campylobacteria, formerly known as Epsilonproteobacteria. These are common primary producers in hydrothermal settings and well established as rTCA 'fixers'. The authors also found a trend of decreasing abundance and diluted 13C enrichment of these fatty acids with distance from the vent system.

This reviewer cannot fault the paper; it's well written and the analytical work appears to be very solid. The only suggestion I offer before the work is published is to include, in the methods section, an explicit statement as to how the FAME values were corrected back to FA for the carbon added during derivatization.

Citation: https://doi.org/10.5194/egusphere-2024-1356-RC1
- AC1: 'Reply on RC1', Joely Maak, 29 Nov 2024
  
  Dear Reviewer,
  We sincerely thank you for your positive evaluation of our manuscript and your kind words about the quality of the writing and analytical work. In the revised manuscript, we will include an explicit statement in the Methods section explaining how the δ¹³C values of fatty acid methyl esters (FAMEs) were corrected back to fatty acids (FA).
  
  Citation: https://doi.org/10.5194/egusphere-2024-1356-AC1
RC2:
'Comment on egusphere-2024-1356', Anonymous Referee #2, 15 Nov 2024

The authors conducted a study on the stable carbon isotope composition of fatty acids from samples collected around shallow-water hydrothermal vents near Kueishantao, Taiwan. Certain fatty acids (C16:1ω7c, C18:1ω9, C18:1ω7c) were notably 13C-enriched and, thus, closer to the measured d13C-DIC values in the water column than other compounds. These fatty acids are known to be produced by sulfur-oxidizing Campylobacteria that are chemoautotrophic microbes using the reductive tricarboxylic acid (rTCA) pathway, which tends to be associated with considerably lower isotopic fractionation during carbon fixation than the Calvin-Benson-Bassham cycle. Consequently, the authors pointed out, the 13C-enriched fatty acids are most likely derived from microbes utilizing the rTCA. The authors further argue that this study provides the first field evidence of lipid synthesis with negligible isotopic fractionation. Their sampling approach, which included sediments, vent fluid, water column particulate organic carbon, and a crab specimen, allowed them to demonstrate a decline in both the abundance and 13C enrichment of these fatty acids with increasing distance from the vent systems.
Overall, this is a well-executed and informative study. I found these shallow-water hydrothermal vents fascinating and appreciated learning about them through this work. I believe that the manuscript could benefit from additional information about the use and limitations of the mass balance model and how potential variations in isotopic fractionation during biosynthesis of different compounds may affect their interpretation. I hope the authors will find the comments below helpful.
-- Is it correct that lipids tend to go through greater isotopic fractionation than other biomolecules, such as proteins and carbohydrates, during biosynthesis? If so, how appropriate it is to draw “hard lines” for rTCA and CBB range of fractionation on Figure 3 and assign specific fatty acids to the closest carbon fixation pathway? Perhaps elaborating on the limitations and uncertainties would be beneficial. Also, would any differences in the extent of isotopic fractionation introduce a level of uncertainty into the mass balance model?
-- Are bulk stable carbon isotopic values available for any of the samples, especially for the crab specimen? It seems like that previous work about this endemic crab used bulk isotopes. I think it would be helpful to report the bulk values (if available) for this crab specimen to make this work more complementary and comparable to previous studies. Especially because the authors propose a different primary food source based on their findings than previous studies (lines 158-159). Here, acknowledging that this study used a single specimen, and consider the likely variability in this omnivorous(?) crab species would be appropriate.
-- The description and interpretation of the mass balance model requires some clarification. This mass balance was used to provide an estimate for the relative abundance of Campylobacteria using lipid biomarkers and their d13C. First, I think it would be fair to the reader to give more information in the Figure 1 caption than just “% Campylobacteria in fatty acids”, I found it confusing. Would calling the axis label “potential Campylobacteria % based on FA proxies” on Fig. 1 plot-D and -G be more appropriate? Moreover, the mass balance is poorly described in the methods. Does it account for isotope values only? Or the relative abundance of the selected lipids is factored into the calculations? My understanding is that only those fatty acids were selected for the mass balance that are known to be synthesized by sulfur-oxidizer Campylobacteria. Wouldn’t such an approach inherently introduce a bias toward Campylobacteria? If yes, wouldn't such statements like the one starting on line 156 would qualify as a circular argument? Line 156-158: "Further, a high lipid content originating from Campylobacteria in the stomach and muscle material (% Campylobacteria: 34% and 32%, Fig. 1) supports a high reliance on carbon derived from rTCA."
-- It took me a while to get an overview of the layout of the sampling stations and understand the sampling design. Personally, I would appreciate more clarity and more visibility about the spatial distribution of the collected samples because it is an important aspect of the study. This issue could be solved by placing a plot-B in the appendix Figure A1 showing the spatial distribution of the collected samples along the transect, including sediment, the crab, and POC. As of now, the reference to see Lin et al for more info is difficult to follow and that study had more stations/sites, so it is not easy to tease out what is applicable here.
-- Perhaps a plot about the sampling layout would also help to place and keep track of all the site acronyms M4, B0…etc. The “My and Mw” were initially called water column samples (Figure 1, 2) than the code names were introduced later (Figure 3) and so on. This approach makes it hard to follow for someone who is not familiar with the spatial distribution of the stations/sites (like me).
-- POC: how many samples and how many liters were filtered?
-- I was curious to check the supporting data to get a better overview of the collected samples, and in the hopes that I may learn something about the mass balance, but it is under moratorium. Although the preprint site says reviewers have access, I couldn’t access it. Also, it seems like it is available through a members-only open access platform that requires personal data to log in (as long as it is in line with BG policy, I have no objection).
-- Given the authors argue it is the first field observation of negligible isotopic fractionation during rTCA, it would be more convincing if they elaborated on why these environmental conditions might result in the observed isotopic effects. Such low pH will certainly shift the DIC dynamics toward CO2 dominance...
Minor comments:
-- line 127: "therefore" should be "we argue" or similar, and the following line has T mentioned twice. Why not call it temperature for clarity?
-- Figure 2 is impossible to interpret in its entirety. Pooling similar sites/samples and show them with the same symbol or using other ways to simplify the figure would help clarity. The point is made that certain FAs exhibit notably 13C-enriched values, but teasing out where they come from is hard to see and interpret.
-- line 142: there is no Figure 4
-- line 153: typo, it should be 'trophic transfer'

Citation: https://doi.org/10.5194/egusphere-2024-1356-RC2
- AC2: 'Reply on RC2', Joely Maak, 29 Nov 2024
  
  Dear Reviewer,
  We sincerely thank you for your thorough review and insightful feedback on our manuscript. We greatly appreciate your time and effort in evaluating our work and providing constructive suggestions. Below, we address each of your points in detail.
  Is it correct that lipids tend to go through greater isotopic fractionation than other biomolecules, such as proteins and carbohydrates, during biosynthesis? If so, how appropriate it is to draw “hard lines” for rTCA and CBB range of fractionation on Figure 3 and assign specific fatty acids to the closest carbon fixation pathway? Perhaps elaborating on the limitations and uncertainties would be beneficial. Also, would any differences in the extent of isotopic fractionation introduce a level of uncertainty into the mass balance model?
  Good point. In the revised manuscript, we will elaborate in the Discussion that lipids undergo greater isotope fractionation compared to other biomolecules (e.g., DeNiro and Epstein 1977, https://doi.org/10.1126/science.327543). To address your concern about the “hard lines” in Figure 2, we will revise the figure caption to explicitly acknowledge the uncertainties in assigning fatty acids to specific pathways. Additionally, we will clarify in the Discussion that while the isotope ranges provide a valuable framework, natural variations in isotope fractionation may introduce uncertainties in using “hard lines”.
  Are bulk stable carbon isotopic values available for any of the samples, especially for the crab specimen? It seems like that previous work about this endemic crab used bulk isotopes. I think it would be helpful to report the bulk values (if available) for this crab specimen to make this work more complementary and comparable to previous studies. Especially because the authors propose a different primary food source based on their findings than previous studies (lines 158-159). Here, acknowledging that this study used a single specimen, and consider the likely variability in this omnivorous(?) crab species would be appropriate.
  Yes, we have bulk stable carbon isotopic values for the crab specimen and will consider the isotope variability of different carbon pools in the crab species.
  The description and interpretation of the mass balance model requires some clarification. This mass balance was used to provide an estimate for the relative abundance of Campylobacteria using lipid biomarkers and their d13C. First, I think it would be fair to the reader to give more information in the Figure 1 caption than just “% Campylobacteria in fatty acids”, I found it confusing. Would calling the axis label “potential Campylobacteria % based on FA proxies” on Fig. 1 plot-D and -G be more appropriate? Moreover, the mass balance is poorly described in the methods. Does it account for isotope values only? Or the relative abundance of the selected lipids is factored into the calculations?
  To clarify, the calculations included the concentration of all detected fatty acids, but only specific fatty acids (C16:1ω7c, C16:1ω7t, n-C16:0, C18:1ω9c, C18:1ω7c, and C18:1ω7t) were assigned to Campylobacteria. The first step involved calculating the percentage of these fatty acids attributable to Campylobacteria based on their δ13C values. Afterward, we calculated the overall percentage of Campylobacteria-related fatty acids as a proportion of the total concentration of all fatty acids detected. We will revise the Methods section to provide a more detailed explanation of this calculation and ensure that readers fully understand the approach.
  My understanding is that only those fatty acids were selected for the mass balance that are known to be synthesized by sulfur-oxidizer Campylobacteria. Wouldn’t such an approach inherently introduce a bias toward Campylobacteria? If yes, wouldn't such statements like the one starting on line 156 would qualify as a circular argument? Line 156-158: "Further, a high lipid content originating from Campylobacteria in the stomach and muscle material (% Campylobacteria: 34% and 32%, Fig. 1) supports a high reliance on carbon derived from rTCA."
  The statement starting on line 156 (“Further, a high lipid content originating from Campylobacteria…”) is not circular because the calculation process is based on a quantitative mass balance that incorporates both δ13C values and the total fatty acid concentrations, rather than selectively considering only those fatty acids synthesized by Campylobacteria. This methodology avoids bias toward Campylobacteria and ensures that the conclusion is supported by the data. To also improve clarity, we will revise the x-axis label in Figure 1D and 1G to "% Campylobacteria based on FA proxies".
  It took me a while to get an overview of the layout of the sampling stations and understand the sampling design. Personally, I would appreciate more clarity and more visibility about the spatial distribution of the collected samples because it is an important aspect of the study. This issue could be solved by placing a plot-B in the appendix Figure A1 showing the spatial distribution of the collected samples along the transect, including sediment, the crab, and POC. As of now, the reference to see Lin et al for more info is difficult to follow and that study had more stations/sites, so it is not easy to tease out what is applicable here.
  Thank you for this suggestion. We will include a new plot in the appendix showing the spatial distribution of the sampling sites, with clear labeling of the sediment and POC collection points. As noted in the preprint, the crab was collected within 30 m of the white vent orifice; unfortunately, more precise coordinates are not available.
  Perhaps a plot about the sampling layout would also help to place and keep track of all the site acronyms M4, B0…etc. The “My and Mw” were initially called water column samples (Figure 1, 2) than the code names were introduced later (Figure 3) and so on. This approach makes it hard to follow for someone who is not familiar with the spatial distribution of the stations/sites (like me).
  We will enhance clarity by including a new plot in the Appendix, accompanied by a table, and ensuring consistent use of acronyms throughout the manuscript.
  POC: how many samples and how many liters were filtered?
  We will include a brief paragraph and a table in the "Experimental Methods" section detailing the POC analysis, including the volume of water filtered for each sample. The table will also provide the sample IDs and their corresponding distances from the vent. In total, 10 POC samples were collected, with filtered volumes ranging from 5 to 18 liters. These samples include one vent fluid sample from each vent, three water column samples collected at distances of 6, 14, and 20 meters from each vent, one sample taken 40 meters from the WV, and a background sample (B0).
  I was curious to check the supporting data to get a better overview of the collected samples, and in the hopes that I may learn something about the mass balance, but it is under moratorium. Although the preprint site says reviewers have access, I couldn’t access it. Also, it seems like it is available through a members-only open access platform that requires personal data to log in (as long as it is in line with BG policy, I have no objection).
  We will send the supporting data to the journal Biogeosciences to make it available to the reviewers. As soon as the manuscript is published it will be openly available.
  Given the authors argue it is the first field observation of negligible isotopic fractionation during rTCA, it would be more convincing if they elaborated on why these environmental conditions might result in the observed isotopic effects. Such low pH will certainly shift the DIC dynamics toward CO2 dominance...
  The reviewer is correct, due to the low pH in the system, CO₂ (dissolved) dominates the DIC pool. Overall, the dissolved inorganic carbon (DIC) concentrations within the vent fluids are very high: 4340 µmol/kg at the white vent and 2450 µmol/kg at the yellow vent. In comparison, samples taken approximately 80 meters from the vent (M80) show concentrations of 2300 µmol/kg (all DIC concentrations from Lin et al., 2019; https://doi.org/10.1016/j.marchem.2019.02.002).
  While the system experiences significant CO₂ degassing, the elevated DIC concentrations are likely sustained by a constant supply of carbon-rich fluids from below. We will expand on this explanation in the manuscript by mentioning “Even though the venting sites are characterized by acidic pH values, which normally shift the DIC dynamics toward CO2 dominance, DIC concentrations at the hydrothermal vents are in excess (Lin et al., 2020).”
  To our knowledge, how and why low pH values or other environmental parameters, such as temperature, affect isotope fractionation has not yet been investigated.
  Minor comments:
  line 127: "therefore" should be "we argue" or similar, and the following line has T mentioned twice. Why not call it temperature for clarity?
  Thanks for pointing that out; we will change it accordingly.
  Figure 2 is impossible to interpret in its entirety. Pooling similar sites/samples and show them with the same symbol or using other ways to simplify the figure would help clarity. The point is made that certain FAs exhibit notably 13C-enriched values, but teasing out where they come from is hard to see and interpret.
  We will simplify Figure 2 by merging the sediment data from the individual venting sites and consolidating the data for Xenograpsus testudinatus. This adjustment will reduce the total number of displayed samples by five.
  line 142: there is no Figure 4
  Changed to Fig. 3
  line 153: typo, it should be 'trophic transfer'
  Done
  
  Citation: https://doi.org/10.5194/egusphere-2024-1356-AC2

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2024-1356', Anonymous Referee #1, 11 Oct 2024

Maak and colleagues report the isotopic compositions of fatty acids in samples collected in the vicinity of a shallow-water hydrothermal system near Kueishantao (Taiwan). The samples comprised particulate organic carbon, sediments and tissues from an endemic crab. While most of the compounds analysed had d¹³C values in the range -23 to -30 per mil consistent with carbon assimilated through CBB pathway, some specific fatty acids namely C16:1ω7c, n-C16:0, C18:1ω7c, were anomalously enriched suggesting they originated from organisms assimilating carbon chemosynthetically through the rTCA pathway. The organisms producing these fatty acids are identified as sulfur oxidising Campylobacteria, formerly known as Epsilonproteobacteria. These are common primary producers in hydrothermal settings and well established as rTCA 'fixers'. The authors also found a trend of decreasing abundance and diluted 13C enrichment of these fatty acids with distance from the vent system.

This reviewer cannot fault the paper; it's well written and the analytical work appears to be very solid. The only suggestion I offer before the work is published is to include, in the methods section, an explicit statement as to how the FAME values were corrected back to FA for the carbon added during derivatization.

Citation: https://doi.org/10.5194/egusphere-2024-1356-RC1
- AC1: 'Reply on RC1', Joely Maak, 29 Nov 2024
  
  Dear Reviewer,
  We sincerely thank you for your positive evaluation of our manuscript and your kind words about the quality of the writing and analytical work. In the revised manuscript, we will include an explicit statement in the Methods section explaining how the δ¹³C values of fatty acid methyl esters (FAMEs) were corrected back to fatty acids (FA).
  
  Citation: https://doi.org/10.5194/egusphere-2024-1356-AC1
RC2:
'Comment on egusphere-2024-1356', Anonymous Referee #2, 15 Nov 2024

The authors conducted a study on the stable carbon isotope composition of fatty acids from samples collected around shallow-water hydrothermal vents near Kueishantao, Taiwan. Certain fatty acids (C16:1ω7c, C18:1ω9, C18:1ω7c) were notably 13C-enriched and, thus, closer to the measured d13C-DIC values in the water column than other compounds. These fatty acids are known to be produced by sulfur-oxidizing Campylobacteria that are chemoautotrophic microbes using the reductive tricarboxylic acid (rTCA) pathway, which tends to be associated with considerably lower isotopic fractionation during carbon fixation than the Calvin-Benson-Bassham cycle. Consequently, the authors pointed out, the 13C-enriched fatty acids are most likely derived from microbes utilizing the rTCA. The authors further argue that this study provides the first field evidence of lipid synthesis with negligible isotopic fractionation. Their sampling approach, which included sediments, vent fluid, water column particulate organic carbon, and a crab specimen, allowed them to demonstrate a decline in both the abundance and 13C enrichment of these fatty acids with increasing distance from the vent systems.
Overall, this is a well-executed and informative study. I found these shallow-water hydrothermal vents fascinating and appreciated learning about them through this work. I believe that the manuscript could benefit from additional information about the use and limitations of the mass balance model and how potential variations in isotopic fractionation during biosynthesis of different compounds may affect their interpretation. I hope the authors will find the comments below helpful.
-- Is it correct that lipids tend to go through greater isotopic fractionation than other biomolecules, such as proteins and carbohydrates, during biosynthesis? If so, how appropriate it is to draw “hard lines” for rTCA and CBB range of fractionation on Figure 3 and assign specific fatty acids to the closest carbon fixation pathway? Perhaps elaborating on the limitations and uncertainties would be beneficial. Also, would any differences in the extent of isotopic fractionation introduce a level of uncertainty into the mass balance model?
-- Are bulk stable carbon isotopic values available for any of the samples, especially for the crab specimen? It seems like that previous work about this endemic crab used bulk isotopes. I think it would be helpful to report the bulk values (if available) for this crab specimen to make this work more complementary and comparable to previous studies. Especially because the authors propose a different primary food source based on their findings than previous studies (lines 158-159). Here, acknowledging that this study used a single specimen, and consider the likely variability in this omnivorous(?) crab species would be appropriate.
-- The description and interpretation of the mass balance model requires some clarification. This mass balance was used to provide an estimate for the relative abundance of Campylobacteria using lipid biomarkers and their d13C. First, I think it would be fair to the reader to give more information in the Figure 1 caption than just “% Campylobacteria in fatty acids”, I found it confusing. Would calling the axis label “potential Campylobacteria % based on FA proxies” on Fig. 1 plot-D and -G be more appropriate? Moreover, the mass balance is poorly described in the methods. Does it account for isotope values only? Or the relative abundance of the selected lipids is factored into the calculations? My understanding is that only those fatty acids were selected for the mass balance that are known to be synthesized by sulfur-oxidizer Campylobacteria. Wouldn’t such an approach inherently introduce a bias toward Campylobacteria? If yes, wouldn't such statements like the one starting on line 156 would qualify as a circular argument? Line 156-158: "Further, a high lipid content originating from Campylobacteria in the stomach and muscle material (% Campylobacteria: 34% and 32%, Fig. 1) supports a high reliance on carbon derived from rTCA."
-- It took me a while to get an overview of the layout of the sampling stations and understand the sampling design. Personally, I would appreciate more clarity and more visibility about the spatial distribution of the collected samples because it is an important aspect of the study. This issue could be solved by placing a plot-B in the appendix Figure A1 showing the spatial distribution of the collected samples along the transect, including sediment, the crab, and POC. As of now, the reference to see Lin et al for more info is difficult to follow and that study had more stations/sites, so it is not easy to tease out what is applicable here.
-- Perhaps a plot about the sampling layout would also help to place and keep track of all the site acronyms M4, B0…etc. The “My and Mw” were initially called water column samples (Figure 1, 2) than the code names were introduced later (Figure 3) and so on. This approach makes it hard to follow for someone who is not familiar with the spatial distribution of the stations/sites (like me).
-- POC: how many samples and how many liters were filtered?
-- I was curious to check the supporting data to get a better overview of the collected samples, and in the hopes that I may learn something about the mass balance, but it is under moratorium. Although the preprint site says reviewers have access, I couldn’t access it. Also, it seems like it is available through a members-only open access platform that requires personal data to log in (as long as it is in line with BG policy, I have no objection).
-- Given the authors argue it is the first field observation of negligible isotopic fractionation during rTCA, it would be more convincing if they elaborated on why these environmental conditions might result in the observed isotopic effects. Such low pH will certainly shift the DIC dynamics toward CO2 dominance...
Minor comments:
-- line 127: "therefore" should be "we argue" or similar, and the following line has T mentioned twice. Why not call it temperature for clarity?
-- Figure 2 is impossible to interpret in its entirety. Pooling similar sites/samples and show them with the same symbol or using other ways to simplify the figure would help clarity. The point is made that certain FAs exhibit notably 13C-enriched values, but teasing out where they come from is hard to see and interpret.
-- line 142: there is no Figure 4
-- line 153: typo, it should be 'trophic transfer'

Citation: https://doi.org/10.5194/egusphere-2024-1356-RC2
- AC2: 'Reply on RC2', Joely Maak, 29 Nov 2024
  
  Dear Reviewer,
  We sincerely thank you for your thorough review and insightful feedback on our manuscript. We greatly appreciate your time and effort in evaluating our work and providing constructive suggestions. Below, we address each of your points in detail.
  Is it correct that lipids tend to go through greater isotopic fractionation than other biomolecules, such as proteins and carbohydrates, during biosynthesis? If so, how appropriate it is to draw “hard lines” for rTCA and CBB range of fractionation on Figure 3 and assign specific fatty acids to the closest carbon fixation pathway? Perhaps elaborating on the limitations and uncertainties would be beneficial. Also, would any differences in the extent of isotopic fractionation introduce a level of uncertainty into the mass balance model?
  Good point. In the revised manuscript, we will elaborate in the Discussion that lipids undergo greater isotope fractionation compared to other biomolecules (e.g., DeNiro and Epstein 1977, https://doi.org/10.1126/science.327543). To address your concern about the “hard lines” in Figure 2, we will revise the figure caption to explicitly acknowledge the uncertainties in assigning fatty acids to specific pathways. Additionally, we will clarify in the Discussion that while the isotope ranges provide a valuable framework, natural variations in isotope fractionation may introduce uncertainties in using “hard lines”.
  Are bulk stable carbon isotopic values available for any of the samples, especially for the crab specimen? It seems like that previous work about this endemic crab used bulk isotopes. I think it would be helpful to report the bulk values (if available) for this crab specimen to make this work more complementary and comparable to previous studies. Especially because the authors propose a different primary food source based on their findings than previous studies (lines 158-159). Here, acknowledging that this study used a single specimen, and consider the likely variability in this omnivorous(?) crab species would be appropriate.
  Yes, we have bulk stable carbon isotopic values for the crab specimen and will consider the isotope variability of different carbon pools in the crab species.
  The description and interpretation of the mass balance model requires some clarification. This mass balance was used to provide an estimate for the relative abundance of Campylobacteria using lipid biomarkers and their d13C. First, I think it would be fair to the reader to give more information in the Figure 1 caption than just “% Campylobacteria in fatty acids”, I found it confusing. Would calling the axis label “potential Campylobacteria % based on FA proxies” on Fig. 1 plot-D and -G be more appropriate? Moreover, the mass balance is poorly described in the methods. Does it account for isotope values only? Or the relative abundance of the selected lipids is factored into the calculations?
  To clarify, the calculations included the concentration of all detected fatty acids, but only specific fatty acids (C16:1ω7c, C16:1ω7t, n-C16:0, C18:1ω9c, C18:1ω7c, and C18:1ω7t) were assigned to Campylobacteria. The first step involved calculating the percentage of these fatty acids attributable to Campylobacteria based on their δ13C values. Afterward, we calculated the overall percentage of Campylobacteria-related fatty acids as a proportion of the total concentration of all fatty acids detected. We will revise the Methods section to provide a more detailed explanation of this calculation and ensure that readers fully understand the approach.
  My understanding is that only those fatty acids were selected for the mass balance that are known to be synthesized by sulfur-oxidizer Campylobacteria. Wouldn’t such an approach inherently introduce a bias toward Campylobacteria? If yes, wouldn't such statements like the one starting on line 156 would qualify as a circular argument? Line 156-158: "Further, a high lipid content originating from Campylobacteria in the stomach and muscle material (% Campylobacteria: 34% and 32%, Fig. 1) supports a high reliance on carbon derived from rTCA."
  The statement starting on line 156 (“Further, a high lipid content originating from Campylobacteria…”) is not circular because the calculation process is based on a quantitative mass balance that incorporates both δ13C values and the total fatty acid concentrations, rather than selectively considering only those fatty acids synthesized by Campylobacteria. This methodology avoids bias toward Campylobacteria and ensures that the conclusion is supported by the data. To also improve clarity, we will revise the x-axis label in Figure 1D and 1G to "% Campylobacteria based on FA proxies".
  It took me a while to get an overview of the layout of the sampling stations and understand the sampling design. Personally, I would appreciate more clarity and more visibility about the spatial distribution of the collected samples because it is an important aspect of the study. This issue could be solved by placing a plot-B in the appendix Figure A1 showing the spatial distribution of the collected samples along the transect, including sediment, the crab, and POC. As of now, the reference to see Lin et al for more info is difficult to follow and that study had more stations/sites, so it is not easy to tease out what is applicable here.
  Thank you for this suggestion. We will include a new plot in the appendix showing the spatial distribution of the sampling sites, with clear labeling of the sediment and POC collection points. As noted in the preprint, the crab was collected within 30 m of the white vent orifice; unfortunately, more precise coordinates are not available.
  Perhaps a plot about the sampling layout would also help to place and keep track of all the site acronyms M4, B0…etc. The “My and Mw” were initially called water column samples (Figure 1, 2) than the code names were introduced later (Figure 3) and so on. This approach makes it hard to follow for someone who is not familiar with the spatial distribution of the stations/sites (like me).
  We will enhance clarity by including a new plot in the Appendix, accompanied by a table, and ensuring consistent use of acronyms throughout the manuscript.
  POC: how many samples and how many liters were filtered?
  We will include a brief paragraph and a table in the "Experimental Methods" section detailing the POC analysis, including the volume of water filtered for each sample. The table will also provide the sample IDs and their corresponding distances from the vent. In total, 10 POC samples were collected, with filtered volumes ranging from 5 to 18 liters. These samples include one vent fluid sample from each vent, three water column samples collected at distances of 6, 14, and 20 meters from each vent, one sample taken 40 meters from the WV, and a background sample (B0).
  I was curious to check the supporting data to get a better overview of the collected samples, and in the hopes that I may learn something about the mass balance, but it is under moratorium. Although the preprint site says reviewers have access, I couldn’t access it. Also, it seems like it is available through a members-only open access platform that requires personal data to log in (as long as it is in line with BG policy, I have no objection).
  We will send the supporting data to the journal Biogeosciences to make it available to the reviewers. As soon as the manuscript is published it will be openly available.
  Given the authors argue it is the first field observation of negligible isotopic fractionation during rTCA, it would be more convincing if they elaborated on why these environmental conditions might result in the observed isotopic effects. Such low pH will certainly shift the DIC dynamics toward CO2 dominance...
  The reviewer is correct, due to the low pH in the system, CO₂ (dissolved) dominates the DIC pool. Overall, the dissolved inorganic carbon (DIC) concentrations within the vent fluids are very high: 4340 µmol/kg at the white vent and 2450 µmol/kg at the yellow vent. In comparison, samples taken approximately 80 meters from the vent (M80) show concentrations of 2300 µmol/kg (all DIC concentrations from Lin et al., 2019; https://doi.org/10.1016/j.marchem.2019.02.002).
  While the system experiences significant CO₂ degassing, the elevated DIC concentrations are likely sustained by a constant supply of carbon-rich fluids from below. We will expand on this explanation in the manuscript by mentioning “Even though the venting sites are characterized by acidic pH values, which normally shift the DIC dynamics toward CO2 dominance, DIC concentrations at the hydrothermal vents are in excess (Lin et al., 2020).”
  To our knowledge, how and why low pH values or other environmental parameters, such as temperature, affect isotope fractionation has not yet been investigated.
  Minor comments:
  line 127: "therefore" should be "we argue" or similar, and the following line has T mentioned twice. Why not call it temperature for clarity?
  Thanks for pointing that out; we will change it accordingly.
  Figure 2 is impossible to interpret in its entirety. Pooling similar sites/samples and show them with the same symbol or using other ways to simplify the figure would help clarity. The point is made that certain FAs exhibit notably 13C-enriched values, but teasing out where they come from is hard to see and interpret.
  We will simplify Figure 2 by merging the sediment data from the individual venting sites and consolidating the data for Xenograpsus testudinatus. This adjustment will reduce the total number of displayed samples by five.
  line 142: there is no Figure 4
  Changed to Fig. 3
  line 153: typo, it should be 'trophic transfer'
  Done
  
  Citation: https://doi.org/10.5194/egusphere-2024-1356-AC2

Peer review completion

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

ED: Reconsider after major revisions (03 Dec 2024) by Andrew Thurber

ED: Reconsider after major revisions (04 Dec 2024) by Sara Vicca (Co-editor-in-chief)

AR by Joely Maak on behalf of the Authors (17 Dec 2024) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (07 Jan 2025) by Andrew Thurber

RR by Anonymous Referee #2 (10 Jan 2025)

ED: Publish as is (17 Jan 2025) by Andrew Thurber

ED: Publish as is (19 Jan 2025) by Paul Stoy (Co-editor-in-chief)

AR by Joely Maak on behalf of the Authors (13 Feb 2025)

Journal article(s) based on this preprint

15 Apr 2025

| Highlight paper

The energy-efficient reductive tricarboxylic acid cycle drives carbon uptake and transfer to higher trophic levels within the Kueishantao shallow-water hydrothermal system

Joely M. Maak, Yu-Shih Lin, Enno Schefuß, Rebecca F. Aepfler, Li-Lian Liu, Marcus Elvert, and Solveig I. Bühring

Biogeosciences, 22, 1853–1863, https://doi.org/10.5194/bg-22-1853-2025,https://doi.org/10.5194/bg-22-1853-2025, 2025

Short summary Co-editor-in-chief

Joely Marie Maak, Yu-Shih Lin, Enno Schefuß, Rebecca F. Aepfler, Li-Lian Liu, Marcus Elvert, and Solveig I. Bühring

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The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.

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In acidic hot springs off Kueishantao, Campylobacteria fix CO₂ by using the reductive tricarboxylic acid cycle (rTCA), causing them to have an isotopically heavier biomass. Here, we showcase extremely low isotopic fractionation (of almost 0 ‰,) which has never been reported in environmental samples. Moreover, the crab Xenograpsus testudinatus relies up to 34 % on Campylobacterial biomass, showcasing the dependency of complex life on microscopic bacteria in harsh environments.


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