the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 29 Sep 2023
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
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.
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Brandon C. Enalls et al.
Status: closed (peer review stopped)
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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
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CC2: 'Reply on CC1', Brandon C Enalls, 05 Oct 2023
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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 -
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
Status: closed (peer review stopped)
-
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
-
CC2: 'Reply on CC1', Brandon C Enalls, 05 Oct 2023
-
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 -
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
Brandon C. Enalls et al.
Brandon C. Enalls et al.
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