the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 05 Oct 2023
Active microbial sulfur cycling in 13,500-year-old lake sediments
Abstract. The addition of sulfur (S) to organic matter to form organosulfur compounds is generally thought to protect organic matter from microbial degradation and promote its preservation. While most microbial sulfur cycling occurs in sulfate-rich sediments above the sulfate-methane transition zone, recently discovered active sulfur cycling in deeper sulfate-poor environments may have a yet-unquantified impact on the mineralization of organic matter. Here we investigated the fate of buried S-compounds down to 10-m sediment depth representing the entire ~13.5 kya history of the sulfate-rich alpine Lake Cadagno. Chemical profiles of sulfate and sulfur reveal that these oxidized species are depleted at the sediment surface with the concomitant formation of iron sulfide minerals. An underlying aquifer provides a second source of sulfate and other oxidants to the deepest and oldest sediment layers generating an inverse redox gradient. At both sulfate depletion zones, isotopes of chromium-reducible sulfur (CRS) and humic-bound sulfur are highly negative (−30 to −65 per mil) compared to background sulfate suggesting ongoing microbial sulfur cycling. Interestingly, humic-bound S from intermittent sediment layers within the sulfate-depleted methanogenesis zone consistently exhibits a lower δ34S than CRS in lacustrine deposits but a higher δ34S than CRS in terrestrial deposits, which could possibly be due to different reactivities of organic matter types (lacustrine versus terrestrial origin) to sulfide or the ability of microorganisms to form/degrade organic S. Although sulfate concentrations are extremely low between the sulfate depletion zones, dsrB gene libraries reveal a huge potential for microbial sulfur reduction throughout the sediment column.
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Status: closed
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RC1: 'Comment on egusphere-2023-2102', Anonymous Referee #1, 06 Nov 2023
Sulfur species in sediments have been used extensively to reconstruct ancient environments, but most of these analyses rely on the canonical assumption that the d34S values preserved in pyrite reflect “sulfide.” Here, the authors present pyrite d34S values from well-constrained, anoxic Lake Cadagno in the context of concentrations and d34S values for other sulfur phases. The observed patterns in near-surface sediments broadly conform to expectations about early microbial sulfate reduction in this environment, but they also include intriguing signals in organic sulfur and in the contrast between sedimentary layer types (lacustrine vs terrestrial). These data are valuable contributions to growing compilations and provide an interesting case study. Still, I was left wanting more in terms of analysis and potential hypotheses for organic S sources, beyond the established (but valid) observation that we lack a real understanding of the mechanisms behind sedimentary sulfur isotopes. I would encourage the authors to consider expanding their analysis to enhance the novelty of the study, prior to eventual publication. The authors should also better address potential sources for high C:S, very low d34S humic acid S values, and the relative abundances of HAS vs. TOS vs. CRS (see below).
Specific comments:
The abundances of organic and inorganic sulfur forms are difficult to compare in their differing units. In Line 220, the text states that humic acid sulfur is the dominant phase present in the deep euxinic sediments, but this species is presented in Fig. 2Ba different unit (mass/mass). The methods indicate that HAS was measured as a sulfur concentration, so why is its molecular weight / mass shown here? Please present the HAS concentrations in a molar S unit (directly measured or calculated from C:S) so it can be more directly compared with the other phases. This becomes especially relevant in the context of Line 237, which appears to make the opposite observation, that most sulfur is inorganic. This balance is a critical aspect of the discussion, so please define these relationships as clearly as possible.
Please also explictly define the relationship between “TOS” used in ratios, kerogen, and humic acid S.
It would increase the future utility of this data to briefly discuss how using a humic acid extraction (versus looking at residual kerogen) might impact comparisons with kerogen data. (Which components of sedimentary organics are included/excluded? Is the HA extraction chemically likely to extract sulfurization products vs reduced vs oxidized organic S species?)
Line 384 – Please lay out your argument more explicitly as to why the observation that specific intervals with very high pyrite concentrations are lacustrine means that most pyrite across the section is authigenic. Not all lacustrine layers show this pattern, what might explain these very high pyrite zones?
Section 4.2 – This discussion should address the abundance of organic S (esp. quantitatively relative to pyrite) alongside its isotope data. If the C:S ratio of HA is representative, it is very high (5,800-13,000), making its very low 34S value especially notable. These very high ratios do also increase the probability that this is an analytical issue – are there sufficient counts on this sulfur peak to be confident in these values? If so, this is a remarkable observation and should be discussed in greater detail.
(How do these C:S ratios compare with other humic acid fractions from terrestrial environments? What does this imply for sulfurization relative to other sources?)
Please note the data types used in Hebting et al. 2006 and Urban 1999 that were used to define early organic matter sulfurization and compare them explicitly with your new results.
Section 4.3 – This section would benefit from an explicit re-statement of how organic S concentration varies across the section in the context of pyrite concentration. There are also some key observations from the data that could be better addressed here.
What sequence of processes do the authors propose to explain the relatively light d34S values in HA lacustrine layers? Why might lacustrine layers differ from each other? Are there alternative explanations for organic S being so extremely light relative to pyrite?
How is sediment remobilization expected to affect pyrite vs organic matter d34S values? How specifically might a shift to more labile organic matter (associated with more lacustrine-type sources) affect these values? How often is that what you observe in the lacustrine-type layers?
The manuscript is motivated in large part by the potential contribution of sulfurization to organic matter preservation, but this is not returned to in the Discussion. How does the data overall provide insights into organic matter preservation? If this is not a primary conclusion, consider de-emphasizing it in the abstract and introduction.
Citation: https://doi.org/10.5194/egusphere-2023-2102-RC1 -
AC1: 'Reply on RC1', Jasmine Berg, 16 Feb 2024
Reviewer 1
The authors have combined an impressive array of various sulfur species and their isotopes, along with distribution of microorganisms and functional genes linked to microbial sulfate reduction and sulfide oxidation to infer sulfur cycling in Lake Cadagno. They found organic-bound S within the sulfate-depleted methanogenesis zone revealed typically low sulfur isotope values compared to the chromium-reducible sulfur, in particular in lacustrine deposits. They argued that the sulfur isotope offset between organic-bound S and chromium-reducible sulfur (mainly pyrite) were likely controlled by the organic matter types. An interesting finding was the accumulation of Fe2+ and Mn2+ in the methanogenic zone. While I find the results interesting, additional discussion and corrections are needed to fully assess the manuscript. Also, additional proper references might be necessary to be inserted in the discussion part.
We have now expanded the discussion to specifically address points on differences between lacustrine and terrestrial sediment deposits as well as 34S signatures in organic-bound versus chromium-reducible sulfur.
- I recommend to reconsider the time when organic-bound S form. The authors listed evidences of the excess free of Fe2+ and Mn2+ in deep euxinic sediment and argued that such environment would favor the formation of metal sulfide, without considering the lack of hydrogen sulfide. I guess most of organic-bound S and pyrite form earlier in shallow sediments (sulfate-sufficient environment) before reaching the current positions.
We have now restructured the discussion to emphasize that the timing likely controls pyritization versus sulfurization as well as isotopic composition. Nonetheless, we would like to point out that the lack of free hydrogen sulfide does not preclude the occurrence of sulfate reduction and therefore organic S or pyrite formation. We have recovered authigenic pyrites from sandy sediments in other environments with no detectable free H2S, though hotspots of sulfate reduction could be proven by inserting Ag-coated photographic film into the sediment for several days (Goodridge and Valentine, 2016).
- The availability of reactive iron in sediment can be a key control on pyrite formation in this study. A limited reactive iron would allow the accumulation of hydrogen sulfide, which further react with organic matter to form organic-bound S. Further discussion on this issue is encouraged (see detailed comments below).
A paragraph has been added to discuss impact of reactive iron availability on pyritization versus sulfurization rates.
- The microbial sulfur cycling in the mediate sulfate-depletion zone (i.e., euxinic zone) seems still active, which is also interesting. But it is also likely that these sulfur-linked microorganisms and functional genes are derived from earlier stages. Consider to combine the geochemical patterns (dissolved sulfate, iron, manganese) to reveal potential biogeochemical processes in this methanic zone.
Since the low amounts of sulfate measured in deep porewaters could also originate from leakage of sulfate-rich lake water during drilling and core retrieval, we remain cautious in interpreting these results. Nonetheless, dsrB gene sequencing reveals that the sulfate-reducing community composition changes in accordance with changing sulfate concentrations rather than simply being preserved during burial. There is also an increase in total dsrB sequence numbers in the deep glacial sediment which was deposited under oxic lake conditions. These microbial communities likely did not derive from earlier stages but rather developed based on sulfate availability at depth. We have now added several lines to the Discussion to compare geochemical conditions (sulfate, iron, manganese) with microbial abundances to conservatively interpret the activity of the dominant microbes there.
- The authors missed up results and discussion to some degree.
The results have been edited to move all interpretation to the discussion as per the specific comments of reviewer 1.
Further specific comments please see blow:
- Line 33: You used the unit per mil here, but ‰ in the main text.
This has been changed to the symbol ‰.
- Lines 34-38: Detailed explanation for this phenomenon in the discussion is lacking.
The reason for observed trends in organic versus pyrite sulfur 34S signatures has now been discussed at length in the Discussion section. Several explanations for these differences could be the origin of sulfur and the timing of organic/inorganic sulfide compound formation.
- Line 56: Light or heavy sulfate are not accurate- you only care sulfur isotopes, but not oxygen. 34S-enriched sulfate and 34S-depleted sulfide are more accurate.
This has been changed to “32S-depleted over heavier 34S-enriched sulfate” for accuracy.
- Lines 58-60: Two parentheses should be avoided.
The parentheses are included in the citations according to the journal specifications. A proof-editor will be able to decide best how to deal with this issue.
- Lines 60: In addition, pyrite can form via polysulfide reaction pathway, or reaction between iron oxides and hydrogen sulfide.
The following has been added to the text: “pyrite (FeS2) which can form via three main pathways: the polysulfide pathway (Rickard, 1975), the H2S pathway (Rickard and Luther, 1997), and the ferric-hydroxide-surface pathway (Wan et al., 2017).”
- Lines 63-64: What do you mean for “porewater fluid”? Please be more specific.
We have now specified “sulfide in porewater fluids”
- Lines 72: See comments above about expression of “heavy and light”.
This line has been changed to : “δ34S-enriched sulfate and δ34S-depleted sulfide”.
- Line 118: When it comes to sulfate-depletion zone (SDZ), I would consider a zone without or with very limited sulfate, like from 20 cm to 780 cm. Suggest to use sulfate-methane transition zone (SMTZ) in your case.
We have chosen to keep the term “sulfate-depletion zone”, which is more accurate for Lake Cadagno sediments. SMTZ implies the (near-)complete removal of methane by sulfate-dependent AOM. This results in only limited overlaps between methane-rich and sulfate-rich sediments - right at the transition from the sulfate-rich zone (sulfate zone) to the methane-rich zone (methane zone). In SMTZs, both sulfate and methane concentrations show clear signs of microbial consumption through a coupled process. Sulfate diffusing from above has a concave-down concentration profile within the SMTZ, methane diffusing up from deeper layers has a concave-up concentration profile within the SMTZ.
Lake Cadagno does not have a clear SMTZ. High sulfate and methane concentrations are present throughout surface sediments. Moreover, while the sulfate concentration profile is concave-down, which indicates microbial sulfate reduction with rates decreasing from the lake floor downward, the methane concentration decreases linearly throughout the sulfate reduction zone (0 to 20 cm) to the lake floor. Sulfate reduction in sediments appears to be mainly controlled by terminal oxidation by electron donors other than methane (e.g. fermentation products), whereas methane oxidation appears to take place mainly at the sediment surface or in the overlying lake water column.
- Lines 155-157: Units for the isotope values of standards (‰) are missing.
We apologize for this omission and have added ‰.
- Lines 160-161: How did you preserve the samples for DNA extraction?
We have now specified that the sediment was frozen. More ample sampling and storage procedures are detailed in Berg et al 2022. In short, samples were taken with a sterile spatula into a sterile cryovial and flash-frozen in the field. Samples were then stored at -80°C until extraction.
- Lines 221-222, 232-234, 238-240, 244-244, 249-251, 257-259: You mixed up results and discussion together. In your framework, these mentioned sentences should go to
“Discussion” part, but not in the “Results”.
These lines have been rephrased or moved to the Discussion except for lines 238-240 which are not an interpretation but a clarification of how the magnitude of TOS impacts calculated ratios. In this case the word ‘interpreted with caution’ is an objective statement.
- Lines 265-266: But you have only a few AVS data at depth.
The wording has been changed in these lines to specify that a comparison was made with the few samples available. “Most samples” was deleted.
- Line: 332: Can you provide the sulfur isotopic composition of evaporite?
The δ34S isotopic composition of evaporite of circa +15‰ has been added to the text.
- Lines 336: Please explain how you excluded AOM in the shallow depth. Both CRS contents and sulfur isotopic composition reveal enrichments in the sulfate depletion zone, which is consistent with AOM. An overlap of sulfate and methane in an interval of 10 cm is commonly observed in typical AOM marine settings.
We have relied on findings from a previous study demonstrating that AOM is present to a very limited degree in the upper 2-3 cm and also at 17 cm depth possibly linked to iron reduction (Schubert et al., 2011; Su et al., 2020). This means that AOM is taking place but is not responsible for the majority of sulfate reduction in the sediment. We have added these two citations and clarified that AOM is likely insignificant, but not absent.
- Lines 338-340: Please be more specific about your findings – “several bacterial genera”.
We have specified these genera are Desulfobulbus, Desulfovibrio, and Desulfomonile.
- Lines 342-345: How can you exclude the limited sulfate was resulted from reoxidation of sulfide (e.g., Treude et al., 2014, GCA; Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: Implications for deep sulfur cycling)? I feel an in-depth discussion on the microbial sulfur cycling in such euxinic mid-column sediments would be much helpful, which is lacking in current shape.
Based on the absence of AVS and excess dissolved Fe2+ and Mn2+, it seems unlikely that there could have been much free sulfide in the mid-column samples. Nonetheless, we acknowledge that this does not exclude the possibility of a cryptic sulfur cycle, with sulfide being reoxidized by metals in the sediment. A new paragraph in the Discussion is in fact dedicated to describing the microbial diversity potentially involved in sulfur cycling, specifically the abundance of dsrB genes belonging to Chloroflexi who have so far not been demonstrated to perform dissimilatory sulfur cycling.
- Line 362: Sulfate limitation might lead to a higher d34S value of sulfide due to the reservoir effect (close system).
To avoid confusion with the Rayleigh effect which might result in 34S-enriched sulfide, we have deleted the word “limitation”.
- Line 366: AOM commonly lead to a high d34S value of sulfide and CRS at the sulfate depletion zone due to the quantitatively conversion of sulfate (e.g., Lin et al., 2016, Chem. Geol.; How sulfate-driven anaerobic oxidation of methane affects the sulfur isotopic composition of pyrite: A SIMS study from the South China Sea).
We thank the reviewer for pointing out this paper and have added this citation.
- Line 373: The terms sulfate reduction coupled to methane oxidation (AOM), anaerobic oxidation of methane (AOM) and sulfate-AOM are used throughout the text. Please keep consistency.
We have changed sulfate reduction coupled to methane oxidation (AOM). Since in line 370 we distinguish between AOM driven by sulfate rather than nitrate, this wording seems to be most appropriate.
- Line 379: Degradation of organic matter might be coupled with iron reduction in this case due to the abundance of iron oxides (e.g., Lovley and Phillips, 1986, Appl. Environ. Microbiol.; Organic matter mineralization with reduction of ferric iron in anaerobic sediments.).
This is definitely true, and discussed along with Mn reduction, fermentation, and other respiration pathways in Berg et al. 2022. Since this paper focuses on sulfur cycling in the Lake Cadagno sediments, we chose not to reiterate more than the pathways directly determining sulfur isotope geochemistry.
- Lines 391-400: How do the differences in organic matter affect the sulfur isotopes? It is not clearly discussed. Although you can see different pattern of sulfur isotopes associated with different kind, but you should also constrain the sulfur isotope pattern of hydrogen sulfide within two environments.
We have now added a description in the Discussion how organic matter quality might affect sulfur isotopes. These are threefold: 1) moieties such as carbonyl groups in labile organic matter react more readily with sulfide which leads to 2) more rapid sulfurization thus preserving more depleted 34S signatures or 3) the additional influence of plant-derived S in the event deposits is more 34S-enriched because plants acquire sulfur via sulfate assimilation with no fractionation.
- Lines 394-396: Please refer to the literature for the C/N ratios, and explain what cause the differences of the C/N ratios.
We have added the following to clarify the relevance of C-N ratios and their origin.
“Mass movement deposits contain more terrestrial plant biomass with C-rich structural compounds while lacustrine deposits are rich in lipids and nitrogenous (Gajendra et al., 2023). The latter are particularly labile due to a combination of relatively weak bonds between monomers and the requirement of nitrogen as a nutrient (Arndt et al., 2013).”
- Line 410: Please note that most of the pyrite might form during early diagenesis at shallow sediment due to the sulfate availability. The increased Fe2+ in mid-column sediments could be due to microbial iron reduction in methanic zones (e.g., Sivan et al., 2011, Limnol. Oceanogr.; Geochemical evidence for iron-mediated anaerobic oxidation of methane). Pyrite found in mid-column sediment has no direct link to the Fe2+ as the lack of sulfide.
We have now modified the Discussion to explain 34S isotope signatures. We ackgnowledge that the main control on isotope signatures are the timing of formation and the source of sulfur.
- Line 414-417: In fact, CRS might represent pyritization over a period of time and late-generated H2S might reveal higher d34S. This might also explain some isotope offset between AVS and CRS
We have changed the discussion to put forward the timing of CRS (and organic-S) formation as the main control on their isotope signatures. Other influencing factors are separately discussed.
- Line 414: How is the Fe2+ availability (vs. hydrogen sulfide) in the experiment of Raven et al. (2021)?
The polysulfide content is 0.5mM. Unfortunately there is no data on iron concentration, simply that iron oxyhydroxides present as discrete, 10–50-μm-diameter particles with a broadly round morphology scattered throughout the marine particles of interest. This does not give any information about Fe:S:C ratios, but the text suggests that since iron hydroxides remained after the experiment, they were not limiting in nature.
- Line 430: The availability of reactive iron can be also an essential control in this case (Liu et al., 2020, GCA). Most of the reactive iron could be consumed at the shallow depth, which limited the further formation of pyrite in depth. This can be supported by the low content of Fe(III). Upon the limited reactive iron, hydrogen sulfide can react with organic matter. Although dissolved Fe2+ was identified in deep depth, this cannot reflect the availability of reactive iron.
We have added a paragraph on the availability of reactive iron in controlling pyritization rates. It is not exactly clear what the reviewer means by the availability of Fe2+ not reflecting the availability of reactive iron. It is true that no measureable Fe2+ was measured in surface sediments, but they did contain oxyhydroxides. The slow release of large amounts of Fe2+ with depth indicates this Fe was reactive. One could also argue that it is the flux of iron and sulfide, rather than concentrations that are important in such environments. There is in fact extensive evidence for the formation of organic S in the presence of iron oxyhydroxides across diverse marine and lacustrine environments (Dale et al., 2009; Filley et al., 2002; Francois, 1987; Hartgers et al., 1997; Raven, Sessions, Fischer, & Adkins, 2016; Urban et al., 1999; van Dongen et al., 2003). In the text we have attempted to reconcile the reviewer’s concerns with different observations presented in the literature.
Citation: https://doi.org/10.5194/egusphere-2023-2102-AC1
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AC1: 'Reply on RC1', Jasmine Berg, 16 Feb 2024
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RC2: 'Comment on egusphere-2023-2102', Anonymous Referee #2, 21 Dec 2023
Review for Manuscript: Active microbial sulfur cycling in 13,500-year-old lake sediments
The authors have combined an impressive array of various sulfur species and their isotopes, along with distribution of microorganisms and functional genes linked to microbial sulfate reduction and sulfide oxidation to infer sulfur cycling in Lake Cadagno. They found organic-bound S within the sulfate-depleted methanogenesis zone revealed typically low sulfur isotope values compared to the chromium-reducible sulfur, in particular in lacustrine deposits. They argued that the sulfur isotope offset between organic-bound S and chromium-reducible sulfur (mainly pyrite) were likely controlled by the organic matter types. An interesting finding was the accumulation of Fe2+ and Mn2+ in the methanogenic zone. While I find the results interesting, additional discussion and corrections are needed to fully assess the manuscript. Also, additional proper references might be necessary to be inserted in the discussion part.
- I recommend to reconsider the time when organic-bound S form. The authors listed evidences of the excess free of Fe2+ and Mn2+ in deep euxinic sediment and argued that such environment would favor the formation of metal sulfide, without considering the lack of hydrogen sulfide. I guess most of organic-bound S and pyrite form earlier in shallow sediments (sulfate-sufficient environment) before reaching the current positions.
- The availability of reactive iron in sediment can be a key control on pyrite formation in this study. A limited reactive iron would allow the accumulation of hydrogen sulfide, which further react with organic matter to form organic-bound S. Further discussion on this issue is encouraged (see detailed comments below).
- The microbial sulfur cycling in the mediate sulfate-depletion zone (i.e., euxinic zone) seems still active, which is also interesting. But it is also likely that these sulfur-linked microorganisms and functional genes are derived from earlier stages. Consider to combine the geochemical patterns (dissolved sulfate, iron, manganese) to reveal potential biogeochemical processes in this methanic zone.
- The authors missed up results and discussion to some degree.
Further specific comments please see blow:
Line 33: You used the unit per mil here, but ‰ in the main text.
Lines 34-38: Detailed explanation for this phenomenon in the discussion is lacking.
Line 56: Light or heavy sulfate are not accurate- you only care sulfur isotopes, but not oxygen. 34S-enriched sulfate and 34S-depleted sulfide are more accurate.
Lines 58-60: Two parentheses should be avoided.
Lines 60: In addition, pyrite can form via polysulfide reaction pathway, or reaction between iron oxides and hydrogen sulfide.
Lines 63-64: What do you mean for “porewater fluid”? Please be more specific.
Lines 72: See comments above about expression of “heavy and light”.
Line 118: When it comes to sulfate-depletion zone (SDZ), I would consider a zone without or with very limited sulfate, like from 20 cm to 780 cm. Suggest to use sulfate-methane transition zone (SMTZ) in your case.
Lines 155-157: Units for the isotope values of standards (‰) are missing.
Lines 160-161: How did you preserve the samples for DNA extraction?
Lines 221-222, 232-234, 238-240, 244-244, 249-251, 257-259: You mixed up results and discussion together. In your framework, these mentioned sentences should go to “Discussion” part, but not in the “Results”.
Lines 265-266: But you have only a few AVS data at depth.
Line: 332: Can you provide the sulfur isotopic composition of evaporite?
Lines 336: Please explain how you excluded AOM in the shallow depth. Both CRS contents and sulfur isotopic composition reveal enrichments in the sulfate depletion zone, which is consistent with AOM. An overlap of sulfate and methane in an interval of 10 cm is commonly observed in typical AOM marine settings.
Lines 338-340: Please be more specific about your findings – “several bacterial genera”.
Lines 342-345: How can you exclude the limited sulfate was resulted from reoxidation of sulfide (e.g., Treude et al., 2014, GCA; Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: Implications for deep sulfur cycling)? I feel an in-depth discussion on the microbial sulfur cycling in such euxinic mid-column sediments would be much helpful, which is lacking in current shape.
Line 362: Sulfate limitation might lead to a higher d34S value of sulfide due to the reservoir effect (close system).
Line 366: AOM commonly lead to a high d34S value of sulfide and CRS at the sulfate depletion zone due to the quantitatively conversion of sulfate (e.g., Lin et al., 2016, Chem. Geol.; How sulfate-driven anaerobic oxidation of methane affects the sulfur isotopic composition of pyrite: A SIMS study from the South China Sea).
Line 373: The terms sulfate reduction coupled to methane oxidation (AOM), anaerobic oxidation of methane (AOM) and sulfate-AOM are used throughout the text. Please keep consistency.
Line 379: Degradation of organic matter might be coupled with iron reduction in this case due to the abundance of iron oxides (e.g., Lovley and Phillips, 1986, Appl. Environ. Microbiol.; Organic matter mineralization with reduction of ferric iron in anaerobic sediments.).
Lines 391-400: How do the differences in organic matter affect the sulfur isotopes? It is not clearly discussed. Although you can see different pattern of sulfur isotopes associated with different kind, but you should also constrain the sulfur isotope pattern of hydrogen sulfide within two environments.
Lines 394-396: Please refer to the literature for the C/N ratios, and explain what cause the differences of the C/N ratios.
Line 410: Please note that most of the pyrite might form during early diagenesis at shallow sediment due to the sulfate availability. The increased Fe2+ in mid-column sediments could be due to microbial iron reduction in methanic zones (e.g., Sivan et al., 2011, Limnol. Oceanogr.; Geochemical evidence for iron-mediated anaerobic oxidation of methane). Pyrite found in mid-column sediment has no direct link to the Fe2+ as the lack of sulfide.
Line 414-417: In fact, CRS might represent pyritization over a period of time and late-generated H2S might reveal higher d34S. This might also explain some isotope offset between AVS and CRS
Line 414: How is the Fe2+ availability (vs. hydrogen sulfide) in the experiment of Raven et al. (2021)?
Line 430: The availability of reactive iron can be also an essential control in this case (Liu et al., 2020, GCA). Most of the reactive iron could be consumed at the shallow depth, which limited the further formation of pyrite in depth. This can be supported by the low content of Fe(III). Upon the limited reactive iron, hydrogen sulfide can react with organic matter. Although dissolved Fe2+ was identified in deep depth, this cannot reflect the availability of reactive iron.
Citation: https://doi.org/10.5194/egusphere-2023-2102-RC2 -
RC3: 'Comment on egusphere-2023-2102', Anonymous Referee #3, 03 Jan 2024
The data on long-term lake sediment is interesting and provides valuable insights into the sulfur cycle and isotopic records in lake sediment. However, I am still unclear about the extremely low d34S value of HAS in lacustrine deposits. The article's logic and content need to be modified. For example, the introduction should clearly state the scientific problem, and discussions should not be included in the results section. Here are some suggestions that may be useful:The standard and instrument used for C, N, and S measurements (Line 138-139) should be introduced, along with the data precision. Additionally, there is no introduction about the measurement of TOC, TS, and TC (see data in Fig.1). It is generally more informative to focus on the ratios of TOC:TS instead of TC vs. TS (Fig. 1, Line 234-235). Alternatively, the inorganic carbon content should be considered.Line 25-26: I’m unclear how this work quantifies the effect of sulfur cycling on the mineralization of organic matter.Line 36-38: The isotope composition of available sulfide should also be considered as a key factor causing variability in humic-bound S.Line 41: At the end of the abstract, the significance or application should be added.Line 54-56: If porewater sulfate concentration is consistent, the sulfur isotope composition of porewater sulfate would also be consistent, even if sulfate reduction occurs. This sentence should be clarified, and a reference should be cited.Line 58: The type and abundance of electron donors are also key factors in controlling sulfur isotope fractionation during microbial sulfate reduction.Line 69-70: It is not right to compare the fractionation factor of a certain process with the isotope composition of an item.Line 79: It is unclear what "isotope fractionation" refers to. Is it the isotope offset between sulfate and pyrite or the isotope fractionation of microbial sulfate reduction? Additionally, it seems that this study is not addressing this particular scientific problem.Line 88: Once again, does this study aim to solve the "scientific problem" of the activity and identity of micro-degradation of organic S pool? After reading this paper, I don’t get much information of micro-degradation of organic S pool. It is advisable to focus on one scientific problem and clearly state your hypothesis.Line 106: I now see your hypothesis, which should have been presented earlier. I suggest reorganizing the introduction section.Line 145: Is DMSO the abbreviation for "Dimethyl sulfoxide"? Why this reagent is added.Caption of Fig.2: Can you provide more details about how the lacustrine deposits are identified?Line 360-362: The isotope composition of CRS and organic S cannot reflect the current sulfur cycle process because it formed early.Line 400: More details are expected to explain how the quality of organic matter affects the sulfur isotope composition of HAS.Line 408-409: The degree of sulfur isotope fractionation during organic matter sulfurization and iron sulfide precipitation should be clarified here.Citation: https://doi.org/
10.5194/egusphere-2023-2102-RC3
Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2023-2102', Anonymous Referee #1, 06 Nov 2023
Sulfur species in sediments have been used extensively to reconstruct ancient environments, but most of these analyses rely on the canonical assumption that the d34S values preserved in pyrite reflect “sulfide.” Here, the authors present pyrite d34S values from well-constrained, anoxic Lake Cadagno in the context of concentrations and d34S values for other sulfur phases. The observed patterns in near-surface sediments broadly conform to expectations about early microbial sulfate reduction in this environment, but they also include intriguing signals in organic sulfur and in the contrast between sedimentary layer types (lacustrine vs terrestrial). These data are valuable contributions to growing compilations and provide an interesting case study. Still, I was left wanting more in terms of analysis and potential hypotheses for organic S sources, beyond the established (but valid) observation that we lack a real understanding of the mechanisms behind sedimentary sulfur isotopes. I would encourage the authors to consider expanding their analysis to enhance the novelty of the study, prior to eventual publication. The authors should also better address potential sources for high C:S, very low d34S humic acid S values, and the relative abundances of HAS vs. TOS vs. CRS (see below).
Specific comments:
The abundances of organic and inorganic sulfur forms are difficult to compare in their differing units. In Line 220, the text states that humic acid sulfur is the dominant phase present in the deep euxinic sediments, but this species is presented in Fig. 2Ba different unit (mass/mass). The methods indicate that HAS was measured as a sulfur concentration, so why is its molecular weight / mass shown here? Please present the HAS concentrations in a molar S unit (directly measured or calculated from C:S) so it can be more directly compared with the other phases. This becomes especially relevant in the context of Line 237, which appears to make the opposite observation, that most sulfur is inorganic. This balance is a critical aspect of the discussion, so please define these relationships as clearly as possible.
Please also explictly define the relationship between “TOS” used in ratios, kerogen, and humic acid S.
It would increase the future utility of this data to briefly discuss how using a humic acid extraction (versus looking at residual kerogen) might impact comparisons with kerogen data. (Which components of sedimentary organics are included/excluded? Is the HA extraction chemically likely to extract sulfurization products vs reduced vs oxidized organic S species?)
Line 384 – Please lay out your argument more explicitly as to why the observation that specific intervals with very high pyrite concentrations are lacustrine means that most pyrite across the section is authigenic. Not all lacustrine layers show this pattern, what might explain these very high pyrite zones?
Section 4.2 – This discussion should address the abundance of organic S (esp. quantitatively relative to pyrite) alongside its isotope data. If the C:S ratio of HA is representative, it is very high (5,800-13,000), making its very low 34S value especially notable. These very high ratios do also increase the probability that this is an analytical issue – are there sufficient counts on this sulfur peak to be confident in these values? If so, this is a remarkable observation and should be discussed in greater detail.
(How do these C:S ratios compare with other humic acid fractions from terrestrial environments? What does this imply for sulfurization relative to other sources?)
Please note the data types used in Hebting et al. 2006 and Urban 1999 that were used to define early organic matter sulfurization and compare them explicitly with your new results.
Section 4.3 – This section would benefit from an explicit re-statement of how organic S concentration varies across the section in the context of pyrite concentration. There are also some key observations from the data that could be better addressed here.
What sequence of processes do the authors propose to explain the relatively light d34S values in HA lacustrine layers? Why might lacustrine layers differ from each other? Are there alternative explanations for organic S being so extremely light relative to pyrite?
How is sediment remobilization expected to affect pyrite vs organic matter d34S values? How specifically might a shift to more labile organic matter (associated with more lacustrine-type sources) affect these values? How often is that what you observe in the lacustrine-type layers?
The manuscript is motivated in large part by the potential contribution of sulfurization to organic matter preservation, but this is not returned to in the Discussion. How does the data overall provide insights into organic matter preservation? If this is not a primary conclusion, consider de-emphasizing it in the abstract and introduction.
Citation: https://doi.org/10.5194/egusphere-2023-2102-RC1 -
AC1: 'Reply on RC1', Jasmine Berg, 16 Feb 2024
Reviewer 1
The authors have combined an impressive array of various sulfur species and their isotopes, along with distribution of microorganisms and functional genes linked to microbial sulfate reduction and sulfide oxidation to infer sulfur cycling in Lake Cadagno. They found organic-bound S within the sulfate-depleted methanogenesis zone revealed typically low sulfur isotope values compared to the chromium-reducible sulfur, in particular in lacustrine deposits. They argued that the sulfur isotope offset between organic-bound S and chromium-reducible sulfur (mainly pyrite) were likely controlled by the organic matter types. An interesting finding was the accumulation of Fe2+ and Mn2+ in the methanogenic zone. While I find the results interesting, additional discussion and corrections are needed to fully assess the manuscript. Also, additional proper references might be necessary to be inserted in the discussion part.
We have now expanded the discussion to specifically address points on differences between lacustrine and terrestrial sediment deposits as well as 34S signatures in organic-bound versus chromium-reducible sulfur.
- I recommend to reconsider the time when organic-bound S form. The authors listed evidences of the excess free of Fe2+ and Mn2+ in deep euxinic sediment and argued that such environment would favor the formation of metal sulfide, without considering the lack of hydrogen sulfide. I guess most of organic-bound S and pyrite form earlier in shallow sediments (sulfate-sufficient environment) before reaching the current positions.
We have now restructured the discussion to emphasize that the timing likely controls pyritization versus sulfurization as well as isotopic composition. Nonetheless, we would like to point out that the lack of free hydrogen sulfide does not preclude the occurrence of sulfate reduction and therefore organic S or pyrite formation. We have recovered authigenic pyrites from sandy sediments in other environments with no detectable free H2S, though hotspots of sulfate reduction could be proven by inserting Ag-coated photographic film into the sediment for several days (Goodridge and Valentine, 2016).
- The availability of reactive iron in sediment can be a key control on pyrite formation in this study. A limited reactive iron would allow the accumulation of hydrogen sulfide, which further react with organic matter to form organic-bound S. Further discussion on this issue is encouraged (see detailed comments below).
A paragraph has been added to discuss impact of reactive iron availability on pyritization versus sulfurization rates.
- The microbial sulfur cycling in the mediate sulfate-depletion zone (i.e., euxinic zone) seems still active, which is also interesting. But it is also likely that these sulfur-linked microorganisms and functional genes are derived from earlier stages. Consider to combine the geochemical patterns (dissolved sulfate, iron, manganese) to reveal potential biogeochemical processes in this methanic zone.
Since the low amounts of sulfate measured in deep porewaters could also originate from leakage of sulfate-rich lake water during drilling and core retrieval, we remain cautious in interpreting these results. Nonetheless, dsrB gene sequencing reveals that the sulfate-reducing community composition changes in accordance with changing sulfate concentrations rather than simply being preserved during burial. There is also an increase in total dsrB sequence numbers in the deep glacial sediment which was deposited under oxic lake conditions. These microbial communities likely did not derive from earlier stages but rather developed based on sulfate availability at depth. We have now added several lines to the Discussion to compare geochemical conditions (sulfate, iron, manganese) with microbial abundances to conservatively interpret the activity of the dominant microbes there.
- The authors missed up results and discussion to some degree.
The results have been edited to move all interpretation to the discussion as per the specific comments of reviewer 1.
Further specific comments please see blow:
- Line 33: You used the unit per mil here, but ‰ in the main text.
This has been changed to the symbol ‰.
- Lines 34-38: Detailed explanation for this phenomenon in the discussion is lacking.
The reason for observed trends in organic versus pyrite sulfur 34S signatures has now been discussed at length in the Discussion section. Several explanations for these differences could be the origin of sulfur and the timing of organic/inorganic sulfide compound formation.
- Line 56: Light or heavy sulfate are not accurate- you only care sulfur isotopes, but not oxygen. 34S-enriched sulfate and 34S-depleted sulfide are more accurate.
This has been changed to “32S-depleted over heavier 34S-enriched sulfate” for accuracy.
- Lines 58-60: Two parentheses should be avoided.
The parentheses are included in the citations according to the journal specifications. A proof-editor will be able to decide best how to deal with this issue.
- Lines 60: In addition, pyrite can form via polysulfide reaction pathway, or reaction between iron oxides and hydrogen sulfide.
The following has been added to the text: “pyrite (FeS2) which can form via three main pathways: the polysulfide pathway (Rickard, 1975), the H2S pathway (Rickard and Luther, 1997), and the ferric-hydroxide-surface pathway (Wan et al., 2017).”
- Lines 63-64: What do you mean for “porewater fluid”? Please be more specific.
We have now specified “sulfide in porewater fluids”
- Lines 72: See comments above about expression of “heavy and light”.
This line has been changed to : “δ34S-enriched sulfate and δ34S-depleted sulfide”.
- Line 118: When it comes to sulfate-depletion zone (SDZ), I would consider a zone without or with very limited sulfate, like from 20 cm to 780 cm. Suggest to use sulfate-methane transition zone (SMTZ) in your case.
We have chosen to keep the term “sulfate-depletion zone”, which is more accurate for Lake Cadagno sediments. SMTZ implies the (near-)complete removal of methane by sulfate-dependent AOM. This results in only limited overlaps between methane-rich and sulfate-rich sediments - right at the transition from the sulfate-rich zone (sulfate zone) to the methane-rich zone (methane zone). In SMTZs, both sulfate and methane concentrations show clear signs of microbial consumption through a coupled process. Sulfate diffusing from above has a concave-down concentration profile within the SMTZ, methane diffusing up from deeper layers has a concave-up concentration profile within the SMTZ.
Lake Cadagno does not have a clear SMTZ. High sulfate and methane concentrations are present throughout surface sediments. Moreover, while the sulfate concentration profile is concave-down, which indicates microbial sulfate reduction with rates decreasing from the lake floor downward, the methane concentration decreases linearly throughout the sulfate reduction zone (0 to 20 cm) to the lake floor. Sulfate reduction in sediments appears to be mainly controlled by terminal oxidation by electron donors other than methane (e.g. fermentation products), whereas methane oxidation appears to take place mainly at the sediment surface or in the overlying lake water column.
- Lines 155-157: Units for the isotope values of standards (‰) are missing.
We apologize for this omission and have added ‰.
- Lines 160-161: How did you preserve the samples for DNA extraction?
We have now specified that the sediment was frozen. More ample sampling and storage procedures are detailed in Berg et al 2022. In short, samples were taken with a sterile spatula into a sterile cryovial and flash-frozen in the field. Samples were then stored at -80°C until extraction.
- Lines 221-222, 232-234, 238-240, 244-244, 249-251, 257-259: You mixed up results and discussion together. In your framework, these mentioned sentences should go to
“Discussion” part, but not in the “Results”.
These lines have been rephrased or moved to the Discussion except for lines 238-240 which are not an interpretation but a clarification of how the magnitude of TOS impacts calculated ratios. In this case the word ‘interpreted with caution’ is an objective statement.
- Lines 265-266: But you have only a few AVS data at depth.
The wording has been changed in these lines to specify that a comparison was made with the few samples available. “Most samples” was deleted.
- Line: 332: Can you provide the sulfur isotopic composition of evaporite?
The δ34S isotopic composition of evaporite of circa +15‰ has been added to the text.
- Lines 336: Please explain how you excluded AOM in the shallow depth. Both CRS contents and sulfur isotopic composition reveal enrichments in the sulfate depletion zone, which is consistent with AOM. An overlap of sulfate and methane in an interval of 10 cm is commonly observed in typical AOM marine settings.
We have relied on findings from a previous study demonstrating that AOM is present to a very limited degree in the upper 2-3 cm and also at 17 cm depth possibly linked to iron reduction (Schubert et al., 2011; Su et al., 2020). This means that AOM is taking place but is not responsible for the majority of sulfate reduction in the sediment. We have added these two citations and clarified that AOM is likely insignificant, but not absent.
- Lines 338-340: Please be more specific about your findings – “several bacterial genera”.
We have specified these genera are Desulfobulbus, Desulfovibrio, and Desulfomonile.
- Lines 342-345: How can you exclude the limited sulfate was resulted from reoxidation of sulfide (e.g., Treude et al., 2014, GCA; Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: Implications for deep sulfur cycling)? I feel an in-depth discussion on the microbial sulfur cycling in such euxinic mid-column sediments would be much helpful, which is lacking in current shape.
Based on the absence of AVS and excess dissolved Fe2+ and Mn2+, it seems unlikely that there could have been much free sulfide in the mid-column samples. Nonetheless, we acknowledge that this does not exclude the possibility of a cryptic sulfur cycle, with sulfide being reoxidized by metals in the sediment. A new paragraph in the Discussion is in fact dedicated to describing the microbial diversity potentially involved in sulfur cycling, specifically the abundance of dsrB genes belonging to Chloroflexi who have so far not been demonstrated to perform dissimilatory sulfur cycling.
- Line 362: Sulfate limitation might lead to a higher d34S value of sulfide due to the reservoir effect (close system).
To avoid confusion with the Rayleigh effect which might result in 34S-enriched sulfide, we have deleted the word “limitation”.
- Line 366: AOM commonly lead to a high d34S value of sulfide and CRS at the sulfate depletion zone due to the quantitatively conversion of sulfate (e.g., Lin et al., 2016, Chem. Geol.; How sulfate-driven anaerobic oxidation of methane affects the sulfur isotopic composition of pyrite: A SIMS study from the South China Sea).
We thank the reviewer for pointing out this paper and have added this citation.
- Line 373: The terms sulfate reduction coupled to methane oxidation (AOM), anaerobic oxidation of methane (AOM) and sulfate-AOM are used throughout the text. Please keep consistency.
We have changed sulfate reduction coupled to methane oxidation (AOM). Since in line 370 we distinguish between AOM driven by sulfate rather than nitrate, this wording seems to be most appropriate.
- Line 379: Degradation of organic matter might be coupled with iron reduction in this case due to the abundance of iron oxides (e.g., Lovley and Phillips, 1986, Appl. Environ. Microbiol.; Organic matter mineralization with reduction of ferric iron in anaerobic sediments.).
This is definitely true, and discussed along with Mn reduction, fermentation, and other respiration pathways in Berg et al. 2022. Since this paper focuses on sulfur cycling in the Lake Cadagno sediments, we chose not to reiterate more than the pathways directly determining sulfur isotope geochemistry.
- Lines 391-400: How do the differences in organic matter affect the sulfur isotopes? It is not clearly discussed. Although you can see different pattern of sulfur isotopes associated with different kind, but you should also constrain the sulfur isotope pattern of hydrogen sulfide within two environments.
We have now added a description in the Discussion how organic matter quality might affect sulfur isotopes. These are threefold: 1) moieties such as carbonyl groups in labile organic matter react more readily with sulfide which leads to 2) more rapid sulfurization thus preserving more depleted 34S signatures or 3) the additional influence of plant-derived S in the event deposits is more 34S-enriched because plants acquire sulfur via sulfate assimilation with no fractionation.
- Lines 394-396: Please refer to the literature for the C/N ratios, and explain what cause the differences of the C/N ratios.
We have added the following to clarify the relevance of C-N ratios and their origin.
“Mass movement deposits contain more terrestrial plant biomass with C-rich structural compounds while lacustrine deposits are rich in lipids and nitrogenous (Gajendra et al., 2023). The latter are particularly labile due to a combination of relatively weak bonds between monomers and the requirement of nitrogen as a nutrient (Arndt et al., 2013).”
- Line 410: Please note that most of the pyrite might form during early diagenesis at shallow sediment due to the sulfate availability. The increased Fe2+ in mid-column sediments could be due to microbial iron reduction in methanic zones (e.g., Sivan et al., 2011, Limnol. Oceanogr.; Geochemical evidence for iron-mediated anaerobic oxidation of methane). Pyrite found in mid-column sediment has no direct link to the Fe2+ as the lack of sulfide.
We have now modified the Discussion to explain 34S isotope signatures. We ackgnowledge that the main control on isotope signatures are the timing of formation and the source of sulfur.
- Line 414-417: In fact, CRS might represent pyritization over a period of time and late-generated H2S might reveal higher d34S. This might also explain some isotope offset between AVS and CRS
We have changed the discussion to put forward the timing of CRS (and organic-S) formation as the main control on their isotope signatures. Other influencing factors are separately discussed.
- Line 414: How is the Fe2+ availability (vs. hydrogen sulfide) in the experiment of Raven et al. (2021)?
The polysulfide content is 0.5mM. Unfortunately there is no data on iron concentration, simply that iron oxyhydroxides present as discrete, 10–50-μm-diameter particles with a broadly round morphology scattered throughout the marine particles of interest. This does not give any information about Fe:S:C ratios, but the text suggests that since iron hydroxides remained after the experiment, they were not limiting in nature.
- Line 430: The availability of reactive iron can be also an essential control in this case (Liu et al., 2020, GCA). Most of the reactive iron could be consumed at the shallow depth, which limited the further formation of pyrite in depth. This can be supported by the low content of Fe(III). Upon the limited reactive iron, hydrogen sulfide can react with organic matter. Although dissolved Fe2+ was identified in deep depth, this cannot reflect the availability of reactive iron.
We have added a paragraph on the availability of reactive iron in controlling pyritization rates. It is not exactly clear what the reviewer means by the availability of Fe2+ not reflecting the availability of reactive iron. It is true that no measureable Fe2+ was measured in surface sediments, but they did contain oxyhydroxides. The slow release of large amounts of Fe2+ with depth indicates this Fe was reactive. One could also argue that it is the flux of iron and sulfide, rather than concentrations that are important in such environments. There is in fact extensive evidence for the formation of organic S in the presence of iron oxyhydroxides across diverse marine and lacustrine environments (Dale et al., 2009; Filley et al., 2002; Francois, 1987; Hartgers et al., 1997; Raven, Sessions, Fischer, & Adkins, 2016; Urban et al., 1999; van Dongen et al., 2003). In the text we have attempted to reconcile the reviewer’s concerns with different observations presented in the literature.
Citation: https://doi.org/10.5194/egusphere-2023-2102-AC1
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AC1: 'Reply on RC1', Jasmine Berg, 16 Feb 2024
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RC2: 'Comment on egusphere-2023-2102', Anonymous Referee #2, 21 Dec 2023
Review for Manuscript: Active microbial sulfur cycling in 13,500-year-old lake sediments
The authors have combined an impressive array of various sulfur species and their isotopes, along with distribution of microorganisms and functional genes linked to microbial sulfate reduction and sulfide oxidation to infer sulfur cycling in Lake Cadagno. They found organic-bound S within the sulfate-depleted methanogenesis zone revealed typically low sulfur isotope values compared to the chromium-reducible sulfur, in particular in lacustrine deposits. They argued that the sulfur isotope offset between organic-bound S and chromium-reducible sulfur (mainly pyrite) were likely controlled by the organic matter types. An interesting finding was the accumulation of Fe2+ and Mn2+ in the methanogenic zone. While I find the results interesting, additional discussion and corrections are needed to fully assess the manuscript. Also, additional proper references might be necessary to be inserted in the discussion part.
- I recommend to reconsider the time when organic-bound S form. The authors listed evidences of the excess free of Fe2+ and Mn2+ in deep euxinic sediment and argued that such environment would favor the formation of metal sulfide, without considering the lack of hydrogen sulfide. I guess most of organic-bound S and pyrite form earlier in shallow sediments (sulfate-sufficient environment) before reaching the current positions.
- The availability of reactive iron in sediment can be a key control on pyrite formation in this study. A limited reactive iron would allow the accumulation of hydrogen sulfide, which further react with organic matter to form organic-bound S. Further discussion on this issue is encouraged (see detailed comments below).
- The microbial sulfur cycling in the mediate sulfate-depletion zone (i.e., euxinic zone) seems still active, which is also interesting. But it is also likely that these sulfur-linked microorganisms and functional genes are derived from earlier stages. Consider to combine the geochemical patterns (dissolved sulfate, iron, manganese) to reveal potential biogeochemical processes in this methanic zone.
- The authors missed up results and discussion to some degree.
Further specific comments please see blow:
Line 33: You used the unit per mil here, but ‰ in the main text.
Lines 34-38: Detailed explanation for this phenomenon in the discussion is lacking.
Line 56: Light or heavy sulfate are not accurate- you only care sulfur isotopes, but not oxygen. 34S-enriched sulfate and 34S-depleted sulfide are more accurate.
Lines 58-60: Two parentheses should be avoided.
Lines 60: In addition, pyrite can form via polysulfide reaction pathway, or reaction between iron oxides and hydrogen sulfide.
Lines 63-64: What do you mean for “porewater fluid”? Please be more specific.
Lines 72: See comments above about expression of “heavy and light”.
Line 118: When it comes to sulfate-depletion zone (SDZ), I would consider a zone without or with very limited sulfate, like from 20 cm to 780 cm. Suggest to use sulfate-methane transition zone (SMTZ) in your case.
Lines 155-157: Units for the isotope values of standards (‰) are missing.
Lines 160-161: How did you preserve the samples for DNA extraction?
Lines 221-222, 232-234, 238-240, 244-244, 249-251, 257-259: You mixed up results and discussion together. In your framework, these mentioned sentences should go to “Discussion” part, but not in the “Results”.
Lines 265-266: But you have only a few AVS data at depth.
Line: 332: Can you provide the sulfur isotopic composition of evaporite?
Lines 336: Please explain how you excluded AOM in the shallow depth. Both CRS contents and sulfur isotopic composition reveal enrichments in the sulfate depletion zone, which is consistent with AOM. An overlap of sulfate and methane in an interval of 10 cm is commonly observed in typical AOM marine settings.
Lines 338-340: Please be more specific about your findings – “several bacterial genera”.
Lines 342-345: How can you exclude the limited sulfate was resulted from reoxidation of sulfide (e.g., Treude et al., 2014, GCA; Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: Implications for deep sulfur cycling)? I feel an in-depth discussion on the microbial sulfur cycling in such euxinic mid-column sediments would be much helpful, which is lacking in current shape.
Line 362: Sulfate limitation might lead to a higher d34S value of sulfide due to the reservoir effect (close system).
Line 366: AOM commonly lead to a high d34S value of sulfide and CRS at the sulfate depletion zone due to the quantitatively conversion of sulfate (e.g., Lin et al., 2016, Chem. Geol.; How sulfate-driven anaerobic oxidation of methane affects the sulfur isotopic composition of pyrite: A SIMS study from the South China Sea).
Line 373: The terms sulfate reduction coupled to methane oxidation (AOM), anaerobic oxidation of methane (AOM) and sulfate-AOM are used throughout the text. Please keep consistency.
Line 379: Degradation of organic matter might be coupled with iron reduction in this case due to the abundance of iron oxides (e.g., Lovley and Phillips, 1986, Appl. Environ. Microbiol.; Organic matter mineralization with reduction of ferric iron in anaerobic sediments.).
Lines 391-400: How do the differences in organic matter affect the sulfur isotopes? It is not clearly discussed. Although you can see different pattern of sulfur isotopes associated with different kind, but you should also constrain the sulfur isotope pattern of hydrogen sulfide within two environments.
Lines 394-396: Please refer to the literature for the C/N ratios, and explain what cause the differences of the C/N ratios.
Line 410: Please note that most of the pyrite might form during early diagenesis at shallow sediment due to the sulfate availability. The increased Fe2+ in mid-column sediments could be due to microbial iron reduction in methanic zones (e.g., Sivan et al., 2011, Limnol. Oceanogr.; Geochemical evidence for iron-mediated anaerobic oxidation of methane). Pyrite found in mid-column sediment has no direct link to the Fe2+ as the lack of sulfide.
Line 414-417: In fact, CRS might represent pyritization over a period of time and late-generated H2S might reveal higher d34S. This might also explain some isotope offset between AVS and CRS
Line 414: How is the Fe2+ availability (vs. hydrogen sulfide) in the experiment of Raven et al. (2021)?
Line 430: The availability of reactive iron can be also an essential control in this case (Liu et al., 2020, GCA). Most of the reactive iron could be consumed at the shallow depth, which limited the further formation of pyrite in depth. This can be supported by the low content of Fe(III). Upon the limited reactive iron, hydrogen sulfide can react with organic matter. Although dissolved Fe2+ was identified in deep depth, this cannot reflect the availability of reactive iron.
Citation: https://doi.org/10.5194/egusphere-2023-2102-RC2 -
RC3: 'Comment on egusphere-2023-2102', Anonymous Referee #3, 03 Jan 2024
The data on long-term lake sediment is interesting and provides valuable insights into the sulfur cycle and isotopic records in lake sediment. However, I am still unclear about the extremely low d34S value of HAS in lacustrine deposits. The article's logic and content need to be modified. For example, the introduction should clearly state the scientific problem, and discussions should not be included in the results section. Here are some suggestions that may be useful:The standard and instrument used for C, N, and S measurements (Line 138-139) should be introduced, along with the data precision. Additionally, there is no introduction about the measurement of TOC, TS, and TC (see data in Fig.1). It is generally more informative to focus on the ratios of TOC:TS instead of TC vs. TS (Fig. 1, Line 234-235). Alternatively, the inorganic carbon content should be considered.Line 25-26: I’m unclear how this work quantifies the effect of sulfur cycling on the mineralization of organic matter.Line 36-38: The isotope composition of available sulfide should also be considered as a key factor causing variability in humic-bound S.Line 41: At the end of the abstract, the significance or application should be added.Line 54-56: If porewater sulfate concentration is consistent, the sulfur isotope composition of porewater sulfate would also be consistent, even if sulfate reduction occurs. This sentence should be clarified, and a reference should be cited.Line 58: The type and abundance of electron donors are also key factors in controlling sulfur isotope fractionation during microbial sulfate reduction.Line 69-70: It is not right to compare the fractionation factor of a certain process with the isotope composition of an item.Line 79: It is unclear what "isotope fractionation" refers to. Is it the isotope offset between sulfate and pyrite or the isotope fractionation of microbial sulfate reduction? Additionally, it seems that this study is not addressing this particular scientific problem.Line 88: Once again, does this study aim to solve the "scientific problem" of the activity and identity of micro-degradation of organic S pool? After reading this paper, I don’t get much information of micro-degradation of organic S pool. It is advisable to focus on one scientific problem and clearly state your hypothesis.Line 106: I now see your hypothesis, which should have been presented earlier. I suggest reorganizing the introduction section.Line 145: Is DMSO the abbreviation for "Dimethyl sulfoxide"? Why this reagent is added.Caption of Fig.2: Can you provide more details about how the lacustrine deposits are identified?Line 360-362: The isotope composition of CRS and organic S cannot reflect the current sulfur cycle process because it formed early.Line 400: More details are expected to explain how the quality of organic matter affects the sulfur isotope composition of HAS.Line 408-409: The degree of sulfur isotope fractionation during organic matter sulfurization and iron sulfide precipitation should be clarified here.Citation: https://doi.org/
10.5194/egusphere-2023-2102-RC3
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