Crystalline Iron Oxides Stimulate Methanogenesis Under Sulfate Reducing Conditions in the Terrestrial Subsurface

Enalls, Brandon C.; Yee, Mon Oo; Bhattacharya, Amrita; Cabugao Grace, Kristine; Gushgari-Doyle, Sara; Hazen, Terry C.; Chakraborty, Romy

doi:https://doi.org/10.5194/bg-2023-174

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https://doi.org/10.5194/bg-2023-174

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29 Sep 2023

| 29 Sep 2023

Status: this preprint was under review for the journal BG but the revision was not accepted.

Crystalline Iron Oxides Stimulate Methanogenesis Under Sulfate Reducing Conditions in the Terrestrial Subsurface

Brandon C. Enalls, Mon Oo Yee, Amrita Bhattacharya, Kristine Cabugao Grace, Sara Gushgari-Doyle, Terry C. Hazen, and Romy Chakraborty

Abstract. Microbial methane production is intimately linked to the biogeochemical cycling of iron, sulfur, and carbon in sedimentary environments. Sulfate-reducing microbes often outcompete methanogens for shared substrates. However, in a prior study at our field site, the Oak Ridge Reservation Field Research Center (ORR FRC) in Oak Ridge, TN, we observed co-occurring sulfate reduction and methanogenesis at 100–150 cm depth where iron (Fe) oxides of varying crystallinities were also detected. Fe oxides are known to act as electron conduits for direct interspecies electron transport (DIET) between syntrophic partners and can connect the metabolisms of methanogens with syntrophic Fe-reducing microbes in nature. However, whether the nature of Fe oxides can influence electron transfer reactions between sulfate-reducing microbes and methanogens is less understood. In this study, we utilized a microbial community enriched from ORR FRC vadose zone sediment to demonstrate the effects of Fe oxides of varying crystallinities on sulfate reduction and methanogenesis. We hypothesized that more crystalline Fe oxides facilitate the co-existence of sulfate-reducers and methanogens. Communities enriched from subsurface sediments produced methane when amended with crystalline hematite but not when amended with the amorphous, short range-ordered (SRO) ferrihydrite. Furthermore, Fe reduction occurred only in incubations amended with SRO ferrihydrite, indicating how poorly crystalline Fe oxides potentially contribute to the dynamic redox nature of the subsurface sediments. Microbial communities enriched during these incubations were composed of several taxa commonly associated with iron and sulfate reduction, fermentation, and methanogenesis, consistent with our geochemical data. Overall, the results from this work deepen our understanding of the role of Fe oxides in extracellular electron transfer, thereby mediating anaerobic metabolisms in the terrestrial subsurface environment.

Received: 20 Sep 2023 – Discussion started: 29 Sep 2023

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Brandon C. Enalls, Mon Oo Yee, Amrita Bhattacharya, Kristine Cabugao Grace, Sara Gushgari-Doyle, Terry C. Hazen, and Romy Chakraborty

Status: closed

CC1:
'Comment on bg-2023-174', David Aromokeye, 01 Oct 2023

This study surely moves the field of DIET forwards and furthers our understanding of the nature of microbe mineral interactions. Previously, ability of crystalline iron oxides to facilitate syntrophic degradation of organic matter by fermenting organisms and methanogens was only shown in ferrugenous settings. The authors demonstrate that during co-existence of sulfare-reducers and methanogens, crystalline iron oxides like hematite can also facilitate enhanced degradation of organic matter. The significance of this study stretches beyond the incubation settings of the study to environments where there is cooccurence of iron and sulfate, such as in coastal sediments with high depositional history where high sediment accumulation distors the typical geochemical zonations such that there is concomitant ebolution of Fe2+ and minor decrease in sulfate concentration in a somewhat cryptic sulfur cycle. Perhaps, crystalline portions mediate more complex microbe interactions in these settings than previously uncovered as this study has shown. My only recommendation to the authors is that their cited the wrong Aromokeye et al, 2020 in their manuscript and should change it appropriately before the paper is published.

Citation: https://doi.org/10.5194/bg-2023-174-CC1
- CC2: 'Reply on CC1', Brandon C Enalls, 05 Oct 2023
  
  Thank you David for your generous comments, we're glad you enjoyed our study. And thank you for pointing out our incorrect citation; we'll be certain to cite the correct study in future versions.
  
  Citation: https://doi.org/10.5194/bg-2023-174-CC2
CC3: 'Comment on bg-2023-174', Liang Shi, 13 Oct 2023

This study investigated the roles of crystalline Fe(III) oxides in direct interspecies electron transfer (DIET) between sulfate-reducing bacteria and methanogens.

L122, butyrate should be an electron donor.
L130, it would be better to compare these two Fe(III) minerals with the same surface area.
L136-139, repetitive?
Materials and Methods, the authors needed to add statistical analyses
Fig. 1, the error bars for sulfate measurement are very large. Why is that?
Fig. 1 SH, the error bars for methane measurement are also very large, which renders the difference between that of hematite treatment and that of others statistical insignificant.
Fig. 4A, the authors need to conduct mass balance analyses to demonstrate that DIET is feasible under the condition tested.

Citation: https://doi.org/10.5194/bg-2023-174-CC3
RC1:
'Comment on bg-2023-174', Anonymous Referee #1, 06 Nov 2023

Dear Authors,
I really liked the idea of your research, especially because the applied methodology together with the scope of the experiments is well suited. However, the writing needs to be improved and the text needs to be spell checked to improve the overall readability.
I stopped the review for now after I have finished reading the geochemical analysis part in 3.1. because I have major concerns about the results you have presented.
You stated in Line 235-236 that your "..control incubations provided context for the changes in analyte concentrations caused by active microbial metabolisms.". Given the experimental design, I would expect no changes in the concentration of butyrate and sulfate in all control incubations and formation of Fe(II) species in incubations with Fe-oxides amended with sulfide due to chemical reduction of Fe(III). However, your analyte concentrations change by more than 20% without reasonable explanation. Further, you explain the loss of 6-7 mM sulfide in controls (Line 230-231) without iron oxides by introducing oxygen while sampling. Assuming just the oxidation of sulfide to elemental sulfur, this equates to the introduction of 3-3.5 mM of molecular oxygen. The oxygen solubility in water/seawater is roughly 300-400 µM. These are huge amounts of oxygen that have to have been present in the headspace and judging by the uniform loss of sulfide in your CS treatments (done in triplicates!), this may very well have been the case in all experiments. Adding such amounts of oxygen will have a huge effect on the biogeochemistry and the microbial community that is adapted to anoxic environments.
Without a reasonable explanation for your observations in the control experiments, I do not think that your results can be published as the potential error of the introduction of oxygen in the sampling procedure, especially in these large amounts, will have dramatic effects on your results in general.

Citation: https://doi.org/10.5194/bg-2023-174-RC1
- AC1: 'Reply on RC1', Brandon C Enalls, 03 Jan 2024
  
  We thank the referee for taking the time to review our manuscript and providing constructive feedback, especially considering our control experiments.
  In regard to decreases in analyte concentrations throughout the experiments, these observations were likely due to variation in handling the samples. We had a change in personnel midway through the incubations and the person that handled the samples from Days 0 and 28 was different from that person that handled Days 57 and 71. Any drops in analyte concentrations between days 28 and 57 were likely due to small differences in the physical amount of sample injected into the Ion Chromatograph and likely do not reflect changes in the concentrations within the cultures themselves.
  All possible controls were in place performing sampling inside anaerobic glove boxes to not introduce ambient oxygen to the headspace of our cultures during sampling. The decreases in sulfide observed in control condition CS over time were likely due to reaction with oxygen in the ambient air post sampling and not in the experimental bottle headspace. Due to unavoidable circumstances due to Covid 19, some time had passed between sampling and the analysis using Cline assay. We took all possible measures to preserve the integrity of our samples by fixing our samples in zinc acetate in order to slow down reactions between oxygen and sulfide, but due to Covid related closures and stipulations in lab space, time did pass between sampling and running the Cline assay and DNA sequencing, Ion Chromatography, XANES analyses, and ferrozine assay at the same time. Personnel changes and lab access stipulations between Days 28 and 57 may partially explain the apparent drops in sulfide between these time points. We edited that section in the manuscript to clarify and reduce ambiguity on what we meant by oxygen exposure during sampling.
  We also reviewed the entire manuscript and checked for spelling and grammar errors as well as edited the material to increase the clarity and readability of the text.
  
  Citation: https://doi.org/10.5194/bg-2023-174-AC1
RC2:
'Comment on bg-2023-174', Anonymous Referee #2, 07 Nov 2023

The paper would be improved by including a broader range of previous studies exploring methanogenesis and sulfate reduction alongside iron oxides, like those by Liu et al. (Bioresource Technology, 2019) and Sivan et al. (PNAS, 2014). The statement in line 89 may be rephrased to reflect prior findings on the coexistence of sulfate-reducing bacteria and methanogenesis with the help of electron transfer with iron oxides.

Due to the use of two sets of primers in the study, it would be clearer to mention them in the figure captions for better clarity. Additionally, the results obtained with the 519F/915R primers seem to have large variability between replicates. Could you address that? The organization of the section on microbial results could also be more coherent; the current presentation seems a bit scattered, particularly when discussing results from the second set of primers. Also: Line 371: “in only a few of our only a few a few”

The paper's discussion on how molybdate affected methanogenesis would benefit from comparison with other research, like Banat et al. (Microbiology, 1983), Tanaka and Lee (Water Science and Technology, 1997), and Isa and Anderson (Process Biochemistry, 2005). Discussing what might lead to the different effects observed in this study would add significant value.

Citation: https://doi.org/10.5194/bg-2023-174-RC2
- AC2: 'Reply on RC2', Brandon C Enalls, 03 Jan 2024
  
  We thank the referee for agreeing to review our manuscript and provide constructive feedback. We have further reviewed the literature on the role of iron oxides on influencing methanogenesis in sulfur (including sources suggested by the referee) containing systems and have reflected this in the latest version of our manuscript.
  We also edited the captions for Figure 3 and Figure S4 to reflect which primer set was used to generate the data presented in the respective figure. It is not surprising to us that there is some heterogeneity in our community composition data. Sedimentary environments are generally known for hosting extremely diverse microbial communities that can vary significantly along small distances at the aggregate scale. The organisms used in our study came from a contaminated and oligotrophic site also known for its steep gradients in physical and chemical parameters, which also likely contributes to any observed heterogeneity. All data is derived from experimental triplicates in an attempt to capture the full diversity of microbes in our samples. In addition, the individual cultures had very low biomass (typically less than 1 ng/uL DNA extracted from each sample) which also could have affected the performance of the primers used for sequencing.
  We further reviewed the literature on the effect of molybdate on methanogenesis (including references suggested by the referee) and have updated our manuscript with more information to support our observations.
  We also reviewed the entire manuscript and checked for spelling and grammar errors as well as edited to increase the clarity of the text.
  
  Citation: https://doi.org/10.5194/bg-2023-174-AC2

Status: closed

CC1:
'Comment on bg-2023-174', David Aromokeye, 01 Oct 2023

This study surely moves the field of DIET forwards and furthers our understanding of the nature of microbe mineral interactions. Previously, ability of crystalline iron oxides to facilitate syntrophic degradation of organic matter by fermenting organisms and methanogens was only shown in ferrugenous settings. The authors demonstrate that during co-existence of sulfare-reducers and methanogens, crystalline iron oxides like hematite can also facilitate enhanced degradation of organic matter. The significance of this study stretches beyond the incubation settings of the study to environments where there is cooccurence of iron and sulfate, such as in coastal sediments with high depositional history where high sediment accumulation distors the typical geochemical zonations such that there is concomitant ebolution of Fe2+ and minor decrease in sulfate concentration in a somewhat cryptic sulfur cycle. Perhaps, crystalline portions mediate more complex microbe interactions in these settings than previously uncovered as this study has shown. My only recommendation to the authors is that their cited the wrong Aromokeye et al, 2020 in their manuscript and should change it appropriately before the paper is published.

Citation: https://doi.org/10.5194/bg-2023-174-CC1
- CC2: 'Reply on CC1', Brandon C Enalls, 05 Oct 2023
  
  Thank you David for your generous comments, we're glad you enjoyed our study. And thank you for pointing out our incorrect citation; we'll be certain to cite the correct study in future versions.
  
  Citation: https://doi.org/10.5194/bg-2023-174-CC2
CC3: 'Comment on bg-2023-174', Liang Shi, 13 Oct 2023

This study investigated the roles of crystalline Fe(III) oxides in direct interspecies electron transfer (DIET) between sulfate-reducing bacteria and methanogens.

L122, butyrate should be an electron donor.
L130, it would be better to compare these two Fe(III) minerals with the same surface area.
L136-139, repetitive?
Materials and Methods, the authors needed to add statistical analyses
Fig. 1, the error bars for sulfate measurement are very large. Why is that?
Fig. 1 SH, the error bars for methane measurement are also very large, which renders the difference between that of hematite treatment and that of others statistical insignificant.
Fig. 4A, the authors need to conduct mass balance analyses to demonstrate that DIET is feasible under the condition tested.

Citation: https://doi.org/10.5194/bg-2023-174-CC3
RC1:
'Comment on bg-2023-174', Anonymous Referee #1, 06 Nov 2023

Dear Authors,
I really liked the idea of your research, especially because the applied methodology together with the scope of the experiments is well suited. However, the writing needs to be improved and the text needs to be spell checked to improve the overall readability.
I stopped the review for now after I have finished reading the geochemical analysis part in 3.1. because I have major concerns about the results you have presented.
You stated in Line 235-236 that your "..control incubations provided context for the changes in analyte concentrations caused by active microbial metabolisms.". Given the experimental design, I would expect no changes in the concentration of butyrate and sulfate in all control incubations and formation of Fe(II) species in incubations with Fe-oxides amended with sulfide due to chemical reduction of Fe(III). However, your analyte concentrations change by more than 20% without reasonable explanation. Further, you explain the loss of 6-7 mM sulfide in controls (Line 230-231) without iron oxides by introducing oxygen while sampling. Assuming just the oxidation of sulfide to elemental sulfur, this equates to the introduction of 3-3.5 mM of molecular oxygen. The oxygen solubility in water/seawater is roughly 300-400 µM. These are huge amounts of oxygen that have to have been present in the headspace and judging by the uniform loss of sulfide in your CS treatments (done in triplicates!), this may very well have been the case in all experiments. Adding such amounts of oxygen will have a huge effect on the biogeochemistry and the microbial community that is adapted to anoxic environments.
Without a reasonable explanation for your observations in the control experiments, I do not think that your results can be published as the potential error of the introduction of oxygen in the sampling procedure, especially in these large amounts, will have dramatic effects on your results in general.

Citation: https://doi.org/10.5194/bg-2023-174-RC1
- AC1: 'Reply on RC1', Brandon C Enalls, 03 Jan 2024
  
  We thank the referee for taking the time to review our manuscript and providing constructive feedback, especially considering our control experiments.
  In regard to decreases in analyte concentrations throughout the experiments, these observations were likely due to variation in handling the samples. We had a change in personnel midway through the incubations and the person that handled the samples from Days 0 and 28 was different from that person that handled Days 57 and 71. Any drops in analyte concentrations between days 28 and 57 were likely due to small differences in the physical amount of sample injected into the Ion Chromatograph and likely do not reflect changes in the concentrations within the cultures themselves.
  All possible controls were in place performing sampling inside anaerobic glove boxes to not introduce ambient oxygen to the headspace of our cultures during sampling. The decreases in sulfide observed in control condition CS over time were likely due to reaction with oxygen in the ambient air post sampling and not in the experimental bottle headspace. Due to unavoidable circumstances due to Covid 19, some time had passed between sampling and the analysis using Cline assay. We took all possible measures to preserve the integrity of our samples by fixing our samples in zinc acetate in order to slow down reactions between oxygen and sulfide, but due to Covid related closures and stipulations in lab space, time did pass between sampling and running the Cline assay and DNA sequencing, Ion Chromatography, XANES analyses, and ferrozine assay at the same time. Personnel changes and lab access stipulations between Days 28 and 57 may partially explain the apparent drops in sulfide between these time points. We edited that section in the manuscript to clarify and reduce ambiguity on what we meant by oxygen exposure during sampling.
  We also reviewed the entire manuscript and checked for spelling and grammar errors as well as edited the material to increase the clarity and readability of the text.
  
  Citation: https://doi.org/10.5194/bg-2023-174-AC1
RC2:
'Comment on bg-2023-174', Anonymous Referee #2, 07 Nov 2023

The paper would be improved by including a broader range of previous studies exploring methanogenesis and sulfate reduction alongside iron oxides, like those by Liu et al. (Bioresource Technology, 2019) and Sivan et al. (PNAS, 2014). The statement in line 89 may be rephrased to reflect prior findings on the coexistence of sulfate-reducing bacteria and methanogenesis with the help of electron transfer with iron oxides.

Due to the use of two sets of primers in the study, it would be clearer to mention them in the figure captions for better clarity. Additionally, the results obtained with the 519F/915R primers seem to have large variability between replicates. Could you address that? The organization of the section on microbial results could also be more coherent; the current presentation seems a bit scattered, particularly when discussing results from the second set of primers. Also: Line 371: “in only a few of our only a few a few”

The paper's discussion on how molybdate affected methanogenesis would benefit from comparison with other research, like Banat et al. (Microbiology, 1983), Tanaka and Lee (Water Science and Technology, 1997), and Isa and Anderson (Process Biochemistry, 2005). Discussing what might lead to the different effects observed in this study would add significant value.

Citation: https://doi.org/10.5194/bg-2023-174-RC2
- AC2: 'Reply on RC2', Brandon C Enalls, 03 Jan 2024
  
  We thank the referee for agreeing to review our manuscript and provide constructive feedback. We have further reviewed the literature on the role of iron oxides on influencing methanogenesis in sulfur (including sources suggested by the referee) containing systems and have reflected this in the latest version of our manuscript.
  We also edited the captions for Figure 3 and Figure S4 to reflect which primer set was used to generate the data presented in the respective figure. It is not surprising to us that there is some heterogeneity in our community composition data. Sedimentary environments are generally known for hosting extremely diverse microbial communities that can vary significantly along small distances at the aggregate scale. The organisms used in our study came from a contaminated and oligotrophic site also known for its steep gradients in physical and chemical parameters, which also likely contributes to any observed heterogeneity. All data is derived from experimental triplicates in an attempt to capture the full diversity of microbes in our samples. In addition, the individual cultures had very low biomass (typically less than 1 ng/uL DNA extracted from each sample) which also could have affected the performance of the primers used for sequencing.
  We further reviewed the literature on the effect of molybdate on methanogenesis (including references suggested by the referee) and have updated our manuscript with more information to support our observations.
  We also reviewed the entire manuscript and checked for spelling and grammar errors as well as edited to increase the clarity of the text.
  
  Citation: https://doi.org/10.5194/bg-2023-174-AC2

Brandon C. Enalls, Mon Oo Yee, Amrita Bhattacharya, Kristine Cabugao Grace, Sara Gushgari-Doyle, Terry C. Hazen, and Romy Chakraborty

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https://doi.org/10.5194/bg-2023-174-supplement

Brandon C. Enalls, Mon Oo Yee, Amrita Bhattacharya, Kristine Cabugao Grace, Sara Gushgari-Doyle, Terry C. Hazen, and Romy Chakraborty

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Short summary

Methanogenic and sulfate-reducing microbes compete for resources in sediments. At our field site, methanogens and sulfate reducers co-exist along with conductive iron oxides, which can transfer electrons between microbes, reducing resource limitation. We incubated microbes with iron oxides of varying crystallinities to test whether they stimulate simultaneous methane production and sulfate reduction. Our results highlight the direct influences mineralogy has on microbial biogeochemistry.


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