the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Down in the dungeons: microbial redox reactions and geochemical transformations define the biogeochemistry of an estuarine sediment column
Abstract. The surface of intertidal estuarine sediments is typically covered with a photosynthetic biofilm. A large fraction of the carbon that is fixed is in the form of exopolymeric substances (EPS), providing the biofilm matrix. The consumption of organic carbon within the sediment column by heterotrophs bacteria is stratified according to the availability of electron acceptors used for organic matter degradation. This sequential use of electron acceptors strongly impacts geochemical gradients and early diagenetic processes within the sediment. In most studies, the distribution and role of the predominant microbial metabolisms is deduced from porewater chemistry and restricted to the upper decimeters of the sediment column, but rarely from direct measurements of microbial activity, potentially leading to erroneous conclusions of biogeochemical processes.
We measured geochemical gradients in three estuarine sediment cores to a depth of 6 meters. Geochemical analyses of porewater and sediment were combined with measurements of microbial activity. In situ microelectrode measurements were performed for pH, oxygen and sulfide. Porewater was extracted and analyzed for major elements using Ion Chromatography, Inductively-Coupled-Plasma, and colorimetric assays for iron speciation. Porewater chemistry was compared to measurements of microbial activity including isothermal calorimetry and metabolic assays (triphenyltetrazolium chloride (TTC) and fluorescein diacetate (FDA)) and concentrations of EPS (sugars, proteins) measured in a previous study on the same cores. Finally, sediment composition was characterized through X-Ray Fluorescence core scanning.
Results show that: (i) aerobic respiration occurred between 0 and 1 cm, (ii) nitrate reduction between 6 and 16 cm, (iii) sulfate reduction between 10 and 50 cm, (iv) manganese oxide reduction between 2–6 and 35–50 cm and (v) iron oxide reduction between 16–18, 24–26 and 35–45 cm. This is concomitant with the area where the microbial activity is the highest. In contrast to the literature, we conclude that some reactions, for example sulfate and nitrate reduction, were locally coupled or at least occurred concomitantly.
Impacts of microbial metabolism on early diagenesis have been modeled via PhreeQc and predicted potential precipitation of metastable iron and/or sulfides. This is confirmed by iron and sulfur increases in sediments characterized through XRF. All these observations have been used to propose a biogeochemical model linking microbial metabolisms and early diagenesis that can be used as a basis for the study of other geochemical profiles in the future.
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RC1: 'Comment on bg-2023-62', Dirk de Beer, 27 Apr 2023
The paper describes a biogeochemical study towards activities in deep estuarine sediments. A broad suite of methods is used, mostly in a correct way. Concluded is that also deep in sediments, below several meters significant activities occur. The discussion lacks depth, not all strange results are recognized, sedimentation time is not considered, nor transport phenomena.
The paper is written in good English, but poor style, making it hard to read. The text in the result section consists of summation of numbers of the plots and table. The text should rather describe the figures and point to the remarkable features, give a first interpretation, that later may be discussed. It is exceptionally difficult reading and not informative. The figures require some basic interpretation for readability, that is missing. The authors should tell us a story, not provide a list of dry facts. Unfortunately, this section must be completely rewritten to make a fluent and readable text.
The summary is good and invites to further reading. The introduction is acceptable but a bit short, focusing on the well-known redox cascade. However, the essential introduction to transport processes in coastal sediments is missing. The rational for the sample location should be described. The location is not ideal, with many complicating influences. The methods is detailed, can be improved at some minor points. The result section is described above. Also in the discussion deep advection, that is often essential for understanding of deep biosphere activities and certainly for close to coast permeable sediments, is missing.
Understanding of transport phenomena, especially in such ‘difficult’ sites is crucial. The site is very difficult, as it is a freshwater site with some marine influence, in a flowing river and very likely with lateral groundwater input. On top of that, the location is in a river bend, with irregular sedimentation.
The figures are made with care, but on several places the letters and numbers must be made larger.
Some details:
From Figure 1 we cannot understand where the 3 cores are taken. Firstly, the 2 maps in 1A should be aligned so that North is on top. Indicated is the location of the box and short core, not of the long core. It is probably not the same spot as the geochemistries are different. The drawings are nice, but the text should be much larger. The depth axes labels are invisibly tiny and of poor quality. I recommend to make more space for the plots. Explain what is a mud pebble and a mud drape. What is the meaning of the letters at the bottom right?
All cores have the label BXN, better remove that as it does not help keeping them apart.
How are the blanks, so the killed controls, treated for the metabolic assays by TTC and FDA?
In Fig 2 the letters and numbers are far too small. Instead of repeating these plots in numbers in the text, describe what we see and what is remarkable. What I find remarkable is the multiple peaks in the sulfate, Fe and Mn plots. Very strange is the high nitrate peak in the sulfidic zone. These observations are important as it is impossible in a 1D interpretation: they can only be result of lateral transport through permeable layers. Thus best is to align these plots with porosity plot or at least the geological info in Fig 1 (mud, sand etc).
In Fig 3 we see again peaks, and nitrate and sulfate correlate: lateral input. Fe2+ is absent where nitrate peaks, nice. The FeIII plots shows impossible concentrations, the method cannot be correct. FeIII does not dissolve well, certainly not to µM levels. Very strange is to have high nitrate levels down to 1.3 m depth. Must be advection and low conversion. Is the land next to the river cultivated and fertilized intensively?
The concentrations of sulfate and Fe differ strongly between the long and short cores. Why?
The paragraph on microbial activities has almost more numbers than text. Unreadable. The hydrolytic activity seems to be hardly decreasing with depth, that is possible, but unlikely. Did you correct for killed controls? Same for the TCC release. Both do not relate with the peak in metabolic activity measured by calorimetry (Table 1). What do you actually measure with TCC release?
The metabolic activity peaks at 50 cm depth, according to table 1. Explain what drives this high rate.
Also the description of the XRF data is sedative. The plots show that the distributions of metals and S correlate. Compare again with the sedimentary log of Fig 1.
The discussion lacks depth. It is a summary of possible microbial conversions. Transport is missing.
L 504 accumulation of nitrate by sulfide is nonsense. Many bacteria oxidise sulfide by nitrate.
L 509 MnO2 driving ammonium oxidiation has been mentioned before, indeed. Interesting, but it does show in the Mn distributions.
L521 Sulfate reduction is very important bit only in the marine realm where sulfate is 28 mM. Here we have very low levels in the order of 100 µM. That is even for rivers very low!
L644 the conceptual model is 1D, right? In the discussion and the model lateral transport is entirely missing. Please check with literature from the waddensea, e.g. by Engelen, Cypionka and Beck on deep biosphere activities in the shallow intertidal flats. See also the review from Joye on transport phenomena and the activities at 20 m depth (doi.org/10.1016/B978-0-444-63893-9.00012-5).
Citation: https://doi.org/10.5194/bg-2023-62-RC1 -
AC1: 'Reply on RC1', Duteil Thibault, 30 Jun 2023
Dear editor,
On behalf of my co-authors, I am submitting a revised version of our manuscript 2023-62 to be considered for publication in Biogeosciences. We have addressed the reviewer’s concerns and questions, either by adding complementary data/observations and/or by justifying our original statements. As requested by reviewer #1, we incorporate the advective transport phenomenon throughout the manuscript, which greatly improved the introduction and discussion sections. We also included additional information about sulfate reduction and methanogenesis, as requested by reviewer #2. Overall, the comments were much appreciated and helped to improve the manuscript. We look forward to your feedback and decision.
Best regards,
Thibault Duteil
General remark to editor and reviewers:
- Four figures (Figures 1, 2, 3 and 10) have been slightly modified compared to the original manuscript and a new figure has been added (Figure 9).
- Parts of the text that were modified or displaced in the original manuscript are written in red.
- Several references recommended by the reviewers or which refine the discussion have been added to the original manuscript.
-Response to Reviewer #1, Dirk De Beer:
The paper describes a biogeochemical study towards activities in deep estuarine sediments. A broad suite of methods is used, mostly in a correct way. Concluded is that also deep in sediments, below several meters significant activities occur. The discussion lacks depth, not all strange results are recognized, sedimentation time is not considered, nor transport phenomena.
-The paper is written in good English, but poor style, making it hard to read. The text in the result section consists of summation of numbers of the plots and table. The text should rather describe the figures and point to the remarkable features, give a first interpretation, that later may be discussed. It is exceptionally difficult reading and not informative. The figures require some basic interpretation for readability, that is missing. The authors should tell us a story, not provide a list of dry facts. Unfortunately, this section must be completely rewritten to make a fluent and readable text
- We understand that this part may require careful reading, but as it contains a large dataset comprising many geochemical, sedimentological and microbial measurements, we feel strongly that this section needs to have a certain level of detail. We prefer to keep most of the original observations (Results) separate from interpretations (Discussion), making results of our study as transparent and clear as possible. However, we took the reviewer’s comment into account and (i) simplified and shortened the Results section in which (ii) we rewrote the part on microbial activity. We trust that this clarifies the results section and improves the readability of the paper.
-The summary is good and invites to further reading. The introduction is acceptable but a bit short, focusing on the well-known redox cascade. However, the essential introduction to transport processes in coastal sediments is missing. The rational for the sample location should be described. The location is not ideal, with many complicating influences. The methods is detailed, can be improved at some minor points. The result section is described above. Also in the discussion deep advection, that is often essential for understanding of deep biosphere activities and certainly for close to coast permeable sediments, is missing.
- We agree with the reviewer’s comments. Consequently, we will expand the introduction and discussion to include transport and lateral advection phenomena, notably to explain the nitrate peak at the surface and the concomitant sulfate and nitrate peaks present at depth. Furthermore, we included two recent references on the topic of transport (Ahmerkamp et al., 2017; Schutte et al. 2019) to improve the discussion of this process. Following the reviewer’s recommendation, the text has been modified in the introduction and the discussion part. We appreciate the reviewer’s comment as it improves the discussion section. We have also specified sample location (Figure 1). The justification for the location of the cores is developed in answer n°3 (see below).
Ahmerkamp, S., Winter, C., Krämer, K., Beer, D. de, Janssen, F., Friedrich, J., Kuypers, M. M. M., and Holtappels, M.: Regulation of benthic oxygen fluxes in permeable sediments of the coastal ocean: Regulation of benthic oxygen fluxes, Limnol. Oceanogr., 62, 1935–1954, https://doi.org/10.1002/lno.10544, 2017.
Schutte, C. A., Ahmerkamp, S., Wu, C. S., Seidel, M., de Beer, D., Cook, P. L. M., and Joye, S. B.: Chapter 12 - Biogeochemical Dynamics of Coastal Tidal Flats, in: Coastal Wetlands (Second Edition), edited by: Perillo, G. M. E., Wolanski, E., Cahoon, D. R., and Hopkinson, C. S., Elsevier, 407–440, https://doi.org/10.1016/B978-0-444-63893-9.00012-5, 2019.
Understanding of transport phenomena, especially in such ‘difficult’ sites is crucial. The site is very difficult, as it is a freshwater site with some marine influence, in a flowing river and very likely with lateral groundwater input. On top of that, the location is in a river bend, with irregular sedimentation.
- We agree with the reviewer’s comment that our site is subject to various surface and underground transport processes and thus complex. The Bordeaux North point bar was selected based on results from previous investigations and remains relatively undisturbed compared to other locations in the Gironde Estuary (e.g., no dredging activity). There is tidal influence present at the site, yet the salinity remains low (between 0.2 and 5 ppt; MAGEST; https://magest.oasu.u-bordeaux.fr/). It is indeed a complex environment where coring is challenging, but we previously drilled three long cores (4-6 meter long), which were studied for sedimentology and petrography (e.g., Virolle et al., 2021). These previous cores allowed to better constrain the point stratigraphic architecture and locate the coring area for this study. Finally, the Bordeaux North point bar is subject to significant sediment deposition and preservation (average sedimentation rate of more than 3 cm.yr-1) since its initiation ca. 300 yrs ago (Virolle et al., 2021). All these reasons justified the choice of this point bar for our study. The hydro-sedimentary context of the study area is now explained in a new section “Hydrological and Sedimentological context” just following the introduction. We think this clarifies the context of the study. Furthermore, the Bordeaux harbor bathymetric maps allowed us to build a cross section linking the sedimentology of the core with the point bar internal stratigraphic architecture (Figure 1; see attached document). The revised version of the paper will include a new sub-section describing this internal architecture and the point bar chronostratigraphy in the results section. We will also add a new figure where geochemical depth profiles and sediment porosity of the Long Core are plotted on this cross section (Figure 9; see attached document). This figure allowed us to support the hypothesis of lateral advection in the permeable horizons of the point bar as suggested by the reviewer, and greatly enhanced the discussion.
MAGEST: https://magest.oasu.u-bordeaux.fr/
Virolle, M., Brigaud, B., Féniès, H., Bourillot, R., Portier, E., Patrier, P., Derriennic, H., and Beaufort, D.: Preservation and distribution of detrital clay coats in a modern estuarine heterolithic point bar in the Gironde estuary (Bordeaux, France), Journal of Sedimentary Research, 91, 812–832, https://doi.org/10.2110/jsr.2020.146, 2021.
The figures are made with care, but on several places the letters and numbers must be made larger.
- We increased the size of the font where it was too small. We think the figures are now more readable thanks for pointing that out.
Some details:
-From Figure 1 we cannot understand where the 3 cores are taken. Firstly, the 2 maps in 1A should be aligned so that North is on top. Indicated is the location of the box and short core, not of the long core. It is probably not the same spot as the geochemistries are different. The drawings are nice, but the text should be much larger. The depth axes labels are invisibly tiny and of poor quality. I recommend to make more space for the plots.
- In the literature, the Gironde estuary is commonly represented as shown in Figure 1 (e.g., Virolle et al., 2021). The position of the long core has been added in the figure as requested and the text in the figure, including that for depth axes, has been enlarged (Figure 1, see attached document).
Virolle, M., Brigaud, B., Féniès, H., Bourillot, R., Portier, E., Patrier, P., Derriennic, H., and Beaufort, D.: Preservation and distribution of detrital clay coats in a modern estuarine heterolithic point bar in the Gironde estuary (Bordeaux, France), Journal of Sedimentary Research, 91, 812–832, https://doi.org/10.2110/jsr.2020.146, 2021.
Explain what is a mud pebble and a mud drape.
- A mud pebble is a centimeter sized rounded particle composed of mud (i.e., a mix of clay and silt-sized particles). Mud pebbles mostly derive from the erosion of low energy intertidal sediments by tidal currents. A mud drape is a centimeter-thick layer of mud deposited at the surface of the dunes and in inter-dune depressions during periods of high and low tide slack water. Both definitions have been added to the Core sedimentary description, sub-section of the Results.
What is the meaning of the letters at the bottom right?
- The bottom letters correspond to the grain size in the sedimentary sections. This is now clarified in the legend of Figures 1 and 2.
-All cores have the label BXN, better remove that as it does not help keeping them apart.
- We agree with the reviewer’s comment and removed the label BXN.
-How are the blanks, so the killed controls, treated for the metabolic assays by TTC and FDA?
- Blanks were prepared by adding 2 mL of 1.5% glutaraldehyde to the sediment and treated in the same manner as live samples. We have added two sentences in the method part to clarify this.
-In Fig 2 the letters and numbers are far too small. Instead of repeating these plots in numbers in the text, describe what we see and what is remarkable. What I find remarkable is the multiple peaks in the sulfate, Fe and Mn plots. Very strange is the high nitrate peak in the sulfidic zone. These observations are important as it is impossible in a 1D interpretation: they can only be result of lateral transport through permeable layers. Thus best is to align these plots with porosity plot or at least the geological info in Fig 1 (mud, sand etc).
- We agree with the reviewer’s comment and added a figure where we align porewater composition and sediment porosity (as measured on thin sections) with the 2D cross section of the point bar (figure 9; see attached document). This figure confirms potential lateral advection of sulfates and nitrates through permeable sand layers, as suggested by the reviewer. This would explain the peaks measured at depth. See answer n°12 below for a detailed response about the nitrate peaks and origin. The discussion has also been expanded, in particular to include potential lateral transport of these solutes through permeable sandy horizons.
-In Fig 3 we see again peaks, and nitrate and sulfate correlate: lateral input. Fe2+ is absent where nitrate peaks, nice. The FeIII plots shows impossible concentrations, the method cannot be correct. FeIII does not dissolve well, certainly not to µM levels.
- We occasionally observed a red precipitate in the Long Core pore water. It is conceivable that there was iron exchange between the colloids and the water, causing this red precipitation. Jones et al. (2011) reported a similar observation in the Satilla River estuary. These authors hypothesized that soluble Fe(III) complexes could be released into the pore waters through reequilibration with the surrounding sediment. We agree that the Fe(III) concentration seems high, however, not impossible. Our values are in the same order of magnitude as reported by e.g., Hopwood et al., 2014, or Beckler et al., 2015 in estuarine porewater. An alternative explanation could be a long term equilibration between porewater and previously precipitated minerals susceptible to release iron, such as iron sulfides. Such minerals would precipitate in the sulfate reduction zone and subsequently dissolve due to slightly more oxidizing conditions deeper in the sediment. Audry et al. (2006) estimated that up to 0.8 mM of iron could be released in the Gironde mud upon iron sulfide dissolution. Beck et al. (2008) also reported concentrations of up to 500 µM of dissolved Fe 5 meters deep in the Wadden Sea intertidal flats. In the revised version of the paper, we will also add measurements of total organic carbon (TOC) in the Long Core sediment (Figure 3; see attached document). We observed a low, but positive correlation between dissolved Fe and TOC, which could indicate the presence of colloidal DOM-Fe complexes in porewater. These different possibilities will be included in the discussion for more clarity. We are grateful for the reviewer’s suggestions to consult the literature on the Wadden Sea which enhanced the discussion.
Audry, S., Blanc, G., Schäfer, J., Chaillou, G., and Robert, S.: Early diagenesis of trace metals (Cd, Cu, Co, Ni, U, Mo, and V) in the freshwater reaches of a macrotidal estuary, Geochimica et Cosmochimica Acta, 70, 2264–2282, https://doi.org/10.1016/j.gca.2006.02.001, 2006.
Beck, M., Dellwig, O., Schnetger, B., and Brumsack, H.-J.: Cycling of trace metals (Mn, Fe, Mo, U, V, Cr) in deep pore waters of intertidal flat sediments, Geochimica et Cosmochimica Acta, 72, 2822–2840, https://doi.org/10.1016/j.gca.2008.04.013, 2008.
Beckler, J. S., Jones, M. E., and Taillefert, M.: The origin, composition, and reactivity of dissolved iron (III) complexes in coastal organic-and iron-rich sediments, Geochimica et Cosmochimica Acta, 152, 72–88, https://doi.org/10.1016/j.gca.2014.12.017, 2015.
Hopwood, M. J., Statham, P. J., and Milani, A.: Dissolved Fe (II) in a river-estuary system rich in dissolved organic matter, Estuarine, Coastal and Shelf Science, 151, 1–9, https://doi.org/10.1016/j.ecss.2014.09.015, 2014.
Jones, M. E., Beckler, J. S., and Taillefert, M.: The flux of soluble organic‐iron (III) complexes from sediments represents a source of stable iron (III) to estuarine waters and to the continental shelf, Limnology and Oceanography, 56, 1811–1823, https://doi.org/10.4319/lo.2011.56.5.1811, 2011.
Very strange is to have high nitrate levels down to 1.3 m depth. Must be advection and low conversion. Is the land next to the river cultivated and fertilized intensively?
- The land abutting the site is an industrial zone. Further upstream as well as downstream from the sampling site are vineyards and land exposed to other agricultural activities. As the reviewer suggests, it is plausible that there are terrestrial inputs from the above-mentioned sources of various nutrients, notably nitrogen and phosphorus (the latter was not measured). We now included in the discussion on lateral flow or, in other words, advective transport (see above) that this could have affected the porewater depth profile of nitrate, and possibly sulfate. It should be noted that the nitrate peak is present in a coarse and permeable sand horizon, corroborating the lateral transport hypothesis (Figure 9; see attached document). The lateral transport (i.e, nitrate flux) can be further modulated by tidal pumping.
-The concentrations of sulfate and Fe differ strongly between the long and short cores. Why?
- Sediment properties vary greatly by location. Even within close proximity there can be a high degree of (lateral) variability, as much as two orders of magnitude, as shown by Charette & Sholkovitz (2006) and Rouxel et al. (2008) in Waquoit Bay (USA) or El Ghobary (1982) and Audry et al. (2006) in the Gironde Estuary. This point has been added to the discussion. Clay layers, as present in Long Core, can be a source for Fe, which could further explain the discrepancy between the Fe values in the two cores.
Audry, S., Blanc, G., Schäfer, J., Chaillou, G., and Robert, S.: Early diagenesis of trace metals (Cd, Cu, Co, Ni, U, Mo, and V) in the freshwater reaches of a macrotidal estuary, Geochimica et Cosmochimica Acta, 70, 2264–2282, https://doi.org/10.1016/j.gca.2006.02.001, 2006.
Charette, M. A. and Sholkovitz, E. R.: Trace element cycling in a subterranean estuary: Part 2. Geochemistry of the pore water, Geochimica et Cosmochimica Acta, 70, 811–826, https://doi.org/10.1016/j.gca.2005.10.019, 2006.
El Ghobary, H.: Interstitial water chemistry of the inner continental shelf sediments off the Gironde Estuary, Estuarine, Coastal and Shelf Science, 16, 639–650, https://doi.org/10.1016/0272-7714(83)90076-8, 1983.
Rouxel, O., Sholkovitz, E., Charette, M., and Edwards, K. J.: Iron isotope fractionation in subterranean estuaries, Geochimica et Cosmochimica Acta, 72, 3413–3430, https://doi.org/10.1016/j.gca.2008.05.001, 2008.
-The paragraph on microbial activities has almost more numbers than text. Unreadable. The hydrolytic activity seems to be hardly decreasing with depth, that is possible, but unlikely. Did you correct for killed controls? Same for the TCC release. Both do not relate with the peak in metabolic activity measured by calorimetry (Table 1). What do you actually measure with TCC release?
- The controls for both TTC and FDA showed zero activity and thus corrections were not necessary. It is common to have different results from these assays and calorimetry. Indeed, although these assays have the same purpose (i.e., measuring metabolic activity), they have rather different targets. The TTC assay is a direct measurement of metabolic activity through reduction of tetrazolium by reductases mostly from the electron transport system (ETS). The FDA assay measures hydrolytic activity of ubiquitous lipases, proteases, and esterases enzymes (i.e., non-specific hydrolases and their capacity to degrade large molecules). We assume that FDA remains high because some of these enzymes (proteases for example) are often quite resistant themselves to degradation once produced; they will continue hydrolyzing their substrate even if the microbial activity has decreased or stopped (Mayer, 1989). While hydrolytic activity (FDA) is positively correlated to TTC (r=0.55; p<0.05; Figure 7), but it shows a strong anticorrelation to porewater pH (r=-0.7; p<0.05; Figure 7). A more acidic pH (ca. 7.9; 0-5 cm) is associated with a more elevated hydrolytic activity, while a higher pH (ca. 8.1-8.2; below 5 cm) is associated with a lower FDA hydrolysis. This point will be added to the discussion of the revised manuscript. The calorimetric assays measure the capacity of the metabolic heat resulting from the activity of microbial community. Isothermal calorimetry contrary to TTC and FDA is not an end-point assay but measures the heatflow in real time, thus providing insight on the time for the microbial community to adapt to the substrate (the lag phase) see next point as well (15). These three assays measure different types of activities but are complementary to each other. They might not yield similar results but show that there are active microbial communities present at different depths albeit with different properties. We will try to make this as clear as possible in the revised version of the manuscript. See detailed review of these assays in Braissant et al., 2020.
Braissant, O., Astasov-Frauenhoffer, M., Waltimo, T., and Bonkat, G.: A review of methods to determine viability, vitality, and metabolic rates in microbiology, Frontiers in Microbiology, 11, 547458, https://doi.org/10.3389/fmicb.2020.547458, 2020.
Mayer, L. M.: Extracellular proteolytic enzyme activity in sediments of an intertidal mudflat, Limnology and Oceanography, 34, 973–981, https://doi.org/10.4319/lo.1989.34.6.0973, 1989.
-The metabolic activity peaks at 50 cm depth, according to table 1. Explain what drives this high rate.
- We will add the value of the surface biofilm to clarify table 1. Generally, the activity tends to decrease with sediment depth, as indicated by the maximum heatflow. There is an even clearer decrease in the heat released Q with depth suggesting that deeper sediments are less prone to support growth (heat being considered as a proxy for the biomass formed or substrate consumed). The sample taken at 50 cm stands out of this overall trend. Indeed, although the microbial community present at 50 cm depth showed a lower heat production compared to surface, it also showed a high activity as well as a rather extended lag phase before consumption of the amended substrate (the higher activity is also supported by the TTC assays that is higher at 50 cm compared to the neighboring samples in the core; Figure 4). This means that the presence of fresh substrate invokes a gene expression in the microbial community that is followed by de novo protein (i.e., enzyme) synthesis. Alternatively, the addition of fresh substrates might trigger spore germination (also relying on the processes described before). Once substrate consumption starts, the activity and the growth rate are indeed higher at 50 cm than in the three other layers. However, the population size at this depth is an order of magnitude smaller than in the other layers. Our CFU counts in this case might not take into account a large proportion of spores from so called strict anaerobes (especially from sulfate reducing bacteria). This is a limitation of our study that must be recognized. In this context, we hypothesize that the germination of spores (from aerobes and anaerobes) as well as the time required for de novo protein (i.e., enzyme) synthesis are responsible for the longer lag time observed (an additional 3 hours compared to all other samples; spore germination is in the order of 150 minutes see ref below). The combined germination of spores in addition to the community present in the sample might result in the high growth rate and activity observed because of the high number of microbes suddenly “reactivated” and growing at the same time. Such hypothesis fits well with the often large proportion of spores observed in sediments (both aerobes and anaerobes; Jorgensen, 2012) and with the time required for a spore to become a growing vegetative cell again (around 150 minutes - see refs below). Also, several spore forming bacteria are known to grow rather fast. Members of the genus Bacillus are good examples, as well as Clostridiales. The origin of those spores cannot be attributed to endogenous or exogenous origin, our observations and the current literature suggest that they could play an important role when substrates become available.
Cupit, C., Lomstein, B. A., and Kjeldsen, K. U.: Contrasting community composition of endospores and vegetative Firmicutes in a marine sediment suggests both endogenous and exogenous sources of endospore accumulation, Environmental Microbiology Reports, 11, 352–360, https://doi.org/10.1111/1758-2229.12679, 2019.
Jørgensen, B. B.: Shrinking majority of the deep biosphere, Proceedings of the National Academy of Sciences, 109, 15976–15977, https://doi.org/10.1073/pnas.12136391, 2012.
O’Sullivan, L. A., Roussel, E. G., Weightman, A. J., Webster, G., Hubert, C. R., Bell, E., Head, I., Sass, H., and Parkes, R. J.: Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure, ISME J, 9, 922–933, https://doi.org/10.1038/ismej.2014.190, 2015.
Sinai, L., Rosenberg, A., Smith, Y., Segev, E., and Ben-Yehuda, S.: The Molecular Timeline of a Reviving Bacterial Spore, Molecular Cell, 57, 695–707, https://doi.org/10.1016/j.molcel.2014.12.019, 2015.
-Also the description of the XRF data is sedative. The plots show that the distributions of metals and S correlate. Compare again with the sedimentary log of Fig 1.
- The distribution of solid metals (Fe, Mn, Al, K) as measured with XRF is largely influenced by the mineralogical nature of the sediments. Clay-rich sediments have relatively high abundance of metals, whereas sands are metal-poor. While we agree that sulfur is correlated with metals in the Box and Short Core, this is not the case in the Long Core. We interpret the parallel increase in sulfur and metal concentrations (as observed in the Box and Short Core between 15 and 40 centimeters) to reflect precipitation of metal-sulfides or clay (Al-S) in the sulfate reduction zone. In Long Core, sulfur and metals sometimes correlate well, and if not, S decrease appears to occur in oxidized zones (especially at greater depth) as indicated by the reddish color of oxidized iron (Fe(III)). Such an oxidized horizon can be observed at 3.5 meters in the long core, where a reddish colors correlates with a decrease of sulfur in XRF (Figure S4).
-The discussion lacks depth. It is a summary of possible microbial conversions. Transport is missing.
- We agree and will add a sub-section on underground water transport (e.g., advection process) in the discussion. As suggested by the reviewer, its potential role on lateral transport of e.g., nitrates in the sediment is now discussed, greatly improving the discussion.
-L 504 accumulation of nitrate by sulfide is nonsense. Many bacteria oxidise sulfide by nitrate.
- We agree with the reviewer that in the absence of oxygen (or under very low oxygen concentrations) sulfide is oxidized with nitrate. However, there are several reports in the literature that even very low sulfide concentrations can inhibit denitrification, notably N2O reduction, in laboratory (Sorensen et al., 1980; Delgado Vela et al., 2018), in brackish and fresh water systems (Aelion & Warttinger, 2010) and in estuaries (Senga et al., 2005). Sulfide may also impact other metabolisms linked with nitrate as for example inhibits Annamox (Carvajal-Arroyo et al., 2013; Jin et al., 2013; Wisniewski et al., 2019). We prefer to include this possibility, but modified the text to indicate that this is a less likely scenario in the discussion part.
Aelion, C. M. and Warttinger, U.: Sulfide inhibition of nitrate removal in coastal sediments, Estuaries and Coasts, 33, 798–803, https://doi.org/10.1007/s12237-010-9275-4, 2010.
Carvajal-Arroyo, J. M., Sun, W., Sierra-Alvarez, R., and Field, J. A.: Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents, Chemosphere, 91, 22–27, https://doi.org/10.1016/j.chemosphere.2012.11.025, 2013.
Delgado Vela, J., Dick, G. J., and Love, N. G.: Sulfide inhibition of nitrite oxidation in activated sludge depends on microbial community composition, Water Research, 138, 241–249, https://doi.org/10.1016/j.watres.2018.03.047, 2018.
Jin, R.-C., Yang, G.-F., Zhang, Q.-Q., Ma, C., Yu, J.-J., and Xing, B.-S.: The effect of sulfide inhibition on the ANAMMOX process, Water Research, 47, 1459–1469, https://doi.org/10.1016/j.watres.2012.12.018, 2013.
Senga, Y., Mochida, K., Fukumori, R., Okamoto, N., and Seike, Y.: N2O accumulation in estuarine and coastal sediments: the influence of H2S on dissimilatory nitrate reduction, Estuarine, Coastal and Shelf Science, 67, 231–238, https://doi.org/10.1016/j.ecss.2005.11.021, 2006.
Sørensen, J., Tiedje, J. M., and Firestone, R. B.: Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens, Applied and Environmental Microbiology, 39, 105–108, https://doi.org/10.1128/aem.39.1.105-108.1980, 1980.
Wisniewski, K., Di Biase, A., Munz, G., Oleszkiewicz, J. A., and Makinia, J.: Kinetic characterization of hydrogen sulfide inhibition of suspended anammox biomass from a membrane bioreactor, Biochemical Engineering Journal, 143, 48–57, https://doi.org/10.1016/j.bej.2018.12.015, 2019.
-L 509 MnO2 driving ammonium oxidation has been mentioned before, indeed. Interesting, but it does show in the Mn distributions.
- The nitrate peak area coincides with an increase in dissolved manganese concentration. Not knowing which hypothesis is the correct one to explain the nitrate peak, we would like to mention all possible scenarios including this one.
-L521 Sulfate reduction is very important bit only in the marine realm where sulfate is 28 mM. Here we have very low levels in the order of 100 µM. That is even for rivers very low!
- As the reviewer points out, the concentration of sulfate is indeed very low (10-100 µM). We report measurements of sulfide in our study, thus reasonably assuming the presence of sulfate reduction (Cypionka et al., 1985; Widdel et al., 1992). Holmkvist et al. (2011) showed sulfate reducing activity below the sulfate-methane transition zone in a nearshore marine sediment despite negligible sulfate concentrations. A recent study by Marietou et al. (2021) reported a km (half saturation constant) for sulfate of 4 µM for a marine sulfate reducing microbe. Based on this, we believe that sulfate reduction can be invoked as one of the microbial metabolisms in our study.
Cypionka, H., Widdel, F., and Pfennig, N.: Survival of sulfate-reducing bacteria after oxygen stress, and growth in sulfate-free oxygen-sulfide gradients, FEMS Microbiology Letters, 31, 39–45, https://doi.org/10.1111/j.1574-6968.1985.tb01129.x, 1985.
Holmkvist, L., Ferdelman, T. G., and Jørgensen, B. B.: A cryptic sulfur cycle driven by iron in the methane zone of marine sediment (Aarhus Bay, Denmark), Geochimica et Cosmochimica Acta, 75, 3581–3599, https://doi.org/10.1016/j.gca.2011.03.033, 2011.
Marietou, A., Kjeldsen, K. U., Glombitza, C., and Jørgensen, B. B.: Response to substrate limitation by a marine sulfate-reducing bacterium, ISME J, 16, 200–210, https://doi.org/10.1038/s41396-021-01061-2, 2022.
Widdel, F. and Bak, F.: Gram-Negative Mesophilic Sulfate-Reducing Bacteria, in: The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, edited by: Balows, A., Trüper, H. G., Dworkin, M., Harder, W., and Schleifer, K.-H., Springer, New York, NY, 3352–3378, https://doi.org/10.1007/978-1-4757-2191-1_21, 1992.
-L644 the conceptual model is 1D, right? In the discussion and the model lateral transport is entirely missing. Please check with literature from the waddensea, e.g. by Engelen, Cypionka and Beck on deep biosphere activities in the shallow intertidal flats. See also the review from Joye on transport phenomena and the activities at 20 m depth (doi.org/10.1016/B978-0-444-63893-9.00012-5).
21. Good remark, we added lateral transport in the model as another manner to explain geochemical gradients measured in this study (notably nitrate and sulfate peaks; Figure 10). In order to improve the model, we will add a 2D sketch at the point bar scale in the model (Fig. 10C). This larger scale sketch will help linking sedimentological and biogeochemical data and to discuss the role of lateral advection. The text describing the model has been updated as well in the discussion part. We would like to thank the reviewer for this relevant comment.
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AC1: 'Reply on RC1', Duteil Thibault, 30 Jun 2023
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RC2: 'Comment on bg-2023-62', Anonymous Referee #2, 15 May 2023
Duteil et al. conducted a series of geochemical and microbiological analyses on sediment cores from the Garonne estuarine channel. Based on porewater and solid-phase geochemical profiles, the authors define the biogeochemical zonation of the sediment cores. Good spatial resolution is achieved in the upper 25 cm, whereas low-resolution profiles of deeper sediments hinder the discussion on certain processes. Microbial activity data are used to facilitate the discussion on biogeochemical processes, but they are not specific to certain processes, which limits their further application. Nevertheless, I found some of the conclusions are justified by the observation and the utilization of metabolic assays and isothermal calorimetry are novel in this type of work. I have several concerns about the interpretation of porewater geochemical profiles as detailed below.
Line 35-37. Given the low resolution of geochemical profiles in the subsurface, I would suggest avoiding defining certain biogeochemical processes to occur at exact depth intervals. For example, it seems to me that iron reduction occurs below 16 cm and manganese reduction occurs below 2 cm to the lower part of the short core. In addition, metal reduction can well be due to abiotic processes involving sulfide oxidation, as the authors mentioned later in the discussion. Therefore, I would suggest interpreting their significance more carefully when discussing their presence in the context of microbial metabolism.
Line 25. “Restricted to the upper decimeters of the sediment column” and “rarely from direct measurements of microbial activity” are not accurate. Porewater geochemical profiles and rates of sulfate reduction and methanogenesis have been frequently measured in both surface and subsurface marine sediments.
Line 96-97. “Potential links between microbial metabolisms and early diagenesis” has been widely studied not only to depths of around 20 cm but also in meter- or kilometer-long sediment cores.
Line 148. The methodology for the analysis of sulfate concentrations and its detection limit is not described. In Figure 3B, the sulfate concentrations are all lower than 12 μM, which is often the detection limit of ion chromatography.
Line 535. Given the extremely low concentrations of sulfate in the subsurface and the low resolution of the sulfate profile below 25 cm, I am not convinced that sulfate reduction occurs below 25 cm in the short core or throughout the long core. My concern is supported by the sulfide profile, in which the sulfide concentrations only increase with depth in the upper 20 cm.
Line 572. Following the comment above, I would instead suggest that methanogenesis occurs from shallow depth (e.g., 25 cm or 1 m) to the deep subsurface. Discrete methanogenic zones are not commonly observed in natural environments.
Citation: https://doi.org/10.5194/bg-2023-62-RC2 -
AC2: 'Reply on RC2', Duteil Thibault, 30 Jun 2023
Dear editor,
On behalf of my co-authors, I am submitting a revised version of our manuscript 2023-62 to be considered for publication in Biogeosciences. We have addressed the reviewer’s concerns and questions, either by adding complementary data/observations and/or by justifying our original statements. As requested by reviewer #1, we incorporate the advective transport phenomenon throughout the manuscript, which greatly improved the introduction and discussion sections. We also included additional information about sulfate reduction and methanogenesis, as requested by reviewer #2. Overall, the comments were much appreciated and helped to improve the manuscript. We look forward to your feedback and decision.
Best regards,
Thibault Duteil
General remark to editor and reviewers:
- Four figures (Figures 1, 2, 3 and 10) have been slightly modified compared to the original manuscript and a new figure has been added (Figure 9).
- Parts of the text that were modified or displaced in the original manuscript are written in red.
- Several references recommended by the reviewers or which refine the discussion have been added to the original manuscript.
Second reviewer:
Duteil et al. conducted a series of geochemical and microbiological analyses on sediment cores from the Garonne estuarine channel. Based on porewater and solid-phase geochemical profiles, the authors define the biogeochemical zonation of the sediment cores. Good spatial resolution is achieved in the upper 25 cm, whereas low-resolution profiles of deeper sediments hinder the discussion on certain processes. Microbial activity data are used to facilitate the discussion on biogeochemical processes, but they are not specific to certain processes, which limits their further application. Nevertheless, I found some of the conclusions are justified by the observation and the utilization of metabolic assays and isothermal calorimetry are novel in this type of work. I have several concerns about the interpretation of porewater geochemical profiles as detailed below.
-Line 35-37. Given the low resolution of geochemical profiles in the subsurface, I would suggest avoiding defining certain biogeochemical processes to occur at exact depth intervals. For example, it seems to me that iron reduction occurs below 16 cm and manganese reduction occurs below 2 cm to the lower part of the short core. In addition, metal reduction can well be due to abiotic processes involving sulfide oxidation, as the authors mentioned later in the discussion. Therefore, I would suggest interpreting their significance more carefully when discussing their presence in the context of microbial metabolism.
22. We appreciate the reviewer’s recommendation. Following his suggestion, we changed the sentence and made more general statements about specific processes without referring to exact depth intervals in the abstract and the discussion parts. We will also be more temperate in the discussion about the conceptual biogeochemical model.
-Line 25. “Restricted to the upper decimeters of the sediment column” and “rarely from direct measurements of microbial activity” are not accurate. Porewater geochemical profiles and rates of sulfate reduction and methanogenesis have been frequently measured in both surface and subsurface marine sediments.
23. Here, we intended to discuss direct measurements of microbial activity, not the rate of metabolic reactions. However, we agree with the reviewer that this sentence is easily misinterpreted. In order to avoid any misunderstanding, we removed the part of lacking microbial measurements from this sentence.
-Line 96-97. “Potential links between microbial metabolisms and early diagenesis” has been widely studied not only to depths of around 20 cm but also in meter- or kilometer-long sediment cores.
24. Per the reviewer’s recommendation, we changed this sentence.
-Line 148. The methodology for the analysis of sulfate concentrations and its detection limit is not described. In Figure 3B, the sulfate concentrations are all lower than 12 μM, which is often the detection limit of ion chromatography.
25. We agree with the reviewer’s comment. The analysis of sulfate should have been included with assay of the other ions, using the ion chromatography. This was corrected in the method section. The detection limit of suppressed ion chromatography is around 0.1 uM, which is lower than the reviewer indicated in this comment. However, we agree with the reviewer that such low concentrations make interpretation of sulfate/sulfide/sulfur cycling complicated and thus we have been more careful when discussing the long core sulfate observations.
-Line 535. Given the extremely low concentrations of sulfate in the subsurface and the low resolution of the sulfate profile below 25 cm, I am not convinced that sulfate reduction occurs below 25 cm in the short core or throughout the long core. My concern is supported by the sulfide profile, in which the sulfide concentrations only increase with depth in the upper 20 cm.
26. We agree with the reviewer that the low spatial resolution associated with analyses of the long core makes it more challenging to identify specific trends in certain metabolisms. The localization of the sulfate reduction in the model (Figure 10) has also been changed. The low sulfate concentrations and decrease with depth (Figures 2 and 3) could, however, be indicative of sulfate reduction. As we mention in the text, in addition to possibly minor reoxidation of sulfide to intermediates other than sulfate (zero-valent sulfur, thiosulfate, polythionates, polysulfides; Visscher & Van Gemerden 1993; Megonigal et al. 2003), sulfides could be associated with metals such as iron (refer to the X-ray fluorescence profiles, Figures 6 and S4). Furthermore (now discussed in much greater detail per Reviewer #1 recommendation), advective (lateral) transport through permeable sandy layers may further distort depth profiles, e.g., locally reoxidizing the sediment, or supplying sulfate or nitrate. For this reason, we carefully described the stratigraphy/architecture by adding a specific sub-section in the results (Point bar architecture and chronostratigraphy) of the sediment column in each of the cores we obtained in order to interpret with care the sulfide data. For further explanations, see our rebuttal on the comment of reviewer 1 (answer 20, about L521) on this topic.
Megonigal, J. P., Hines, M. E., and Visscher, P. T.: Anaerobic metabolism: linkages to trace gases and aerobic processes, Biogeochemistry, 8, 317–424, https://doi.org/10.1016/B0-08-043751-6/08132-9, 2003.
Visscher, P. T. and Van Gemerden: Sulfur Cycling in Laminated Marine Microbial Ecosystems | SpringerLink, 1993.
-Line 572. Following the comment above, I would instead suggest that methanogenesis occurs from shallow depth (e.g., 25 cm or 1 m) to the deep subsurface. Discrete methanogenic zones are not commonly observed in natural environments.
27. We agree with the reviewer that typically, methanogenesis below the sulfate-methane transition zone is continuous and methane concentrations (not measured in our study), should increase with depth (Megonigal et al. 2003). However, as shown in Figure 3, for several reasons (e.g., rapid burial of sediments, lateral advection in coarse sand layers) the organic carbon distribution is heterogeneous. It is therefore conceivable, with some care given the larger intervals of which we made observations, that there are certain sedimentary horizons associated with higher carbon contents that support higher rates of methanogenesis. To support this hypothesis, we have added the concentrations of total organic carbon (TOC) in the long core (Figure 3; see attached document). We will also modify the discussion to include TOC data for a more in-depth discussion on this aspect.
Megonigal, J. P., Hines, M. E., and Visscher, P. T.: Anaerobic metabolism: linkages to trace gases and aerobic processes, Biogeochemistry, 8, 317–424, https://doi.org/10.1016/B0-08-043751-6/08132-9, 2003.
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AC2: 'Reply on RC2', Duteil Thibault, 30 Jun 2023
Status: closed
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RC1: 'Comment on bg-2023-62', Dirk de Beer, 27 Apr 2023
The paper describes a biogeochemical study towards activities in deep estuarine sediments. A broad suite of methods is used, mostly in a correct way. Concluded is that also deep in sediments, below several meters significant activities occur. The discussion lacks depth, not all strange results are recognized, sedimentation time is not considered, nor transport phenomena.
The paper is written in good English, but poor style, making it hard to read. The text in the result section consists of summation of numbers of the plots and table. The text should rather describe the figures and point to the remarkable features, give a first interpretation, that later may be discussed. It is exceptionally difficult reading and not informative. The figures require some basic interpretation for readability, that is missing. The authors should tell us a story, not provide a list of dry facts. Unfortunately, this section must be completely rewritten to make a fluent and readable text.
The summary is good and invites to further reading. The introduction is acceptable but a bit short, focusing on the well-known redox cascade. However, the essential introduction to transport processes in coastal sediments is missing. The rational for the sample location should be described. The location is not ideal, with many complicating influences. The methods is detailed, can be improved at some minor points. The result section is described above. Also in the discussion deep advection, that is often essential for understanding of deep biosphere activities and certainly for close to coast permeable sediments, is missing.
Understanding of transport phenomena, especially in such ‘difficult’ sites is crucial. The site is very difficult, as it is a freshwater site with some marine influence, in a flowing river and very likely with lateral groundwater input. On top of that, the location is in a river bend, with irregular sedimentation.
The figures are made with care, but on several places the letters and numbers must be made larger.
Some details:
From Figure 1 we cannot understand where the 3 cores are taken. Firstly, the 2 maps in 1A should be aligned so that North is on top. Indicated is the location of the box and short core, not of the long core. It is probably not the same spot as the geochemistries are different. The drawings are nice, but the text should be much larger. The depth axes labels are invisibly tiny and of poor quality. I recommend to make more space for the plots. Explain what is a mud pebble and a mud drape. What is the meaning of the letters at the bottom right?
All cores have the label BXN, better remove that as it does not help keeping them apart.
How are the blanks, so the killed controls, treated for the metabolic assays by TTC and FDA?
In Fig 2 the letters and numbers are far too small. Instead of repeating these plots in numbers in the text, describe what we see and what is remarkable. What I find remarkable is the multiple peaks in the sulfate, Fe and Mn plots. Very strange is the high nitrate peak in the sulfidic zone. These observations are important as it is impossible in a 1D interpretation: they can only be result of lateral transport through permeable layers. Thus best is to align these plots with porosity plot or at least the geological info in Fig 1 (mud, sand etc).
In Fig 3 we see again peaks, and nitrate and sulfate correlate: lateral input. Fe2+ is absent where nitrate peaks, nice. The FeIII plots shows impossible concentrations, the method cannot be correct. FeIII does not dissolve well, certainly not to µM levels. Very strange is to have high nitrate levels down to 1.3 m depth. Must be advection and low conversion. Is the land next to the river cultivated and fertilized intensively?
The concentrations of sulfate and Fe differ strongly between the long and short cores. Why?
The paragraph on microbial activities has almost more numbers than text. Unreadable. The hydrolytic activity seems to be hardly decreasing with depth, that is possible, but unlikely. Did you correct for killed controls? Same for the TCC release. Both do not relate with the peak in metabolic activity measured by calorimetry (Table 1). What do you actually measure with TCC release?
The metabolic activity peaks at 50 cm depth, according to table 1. Explain what drives this high rate.
Also the description of the XRF data is sedative. The plots show that the distributions of metals and S correlate. Compare again with the sedimentary log of Fig 1.
The discussion lacks depth. It is a summary of possible microbial conversions. Transport is missing.
L 504 accumulation of nitrate by sulfide is nonsense. Many bacteria oxidise sulfide by nitrate.
L 509 MnO2 driving ammonium oxidiation has been mentioned before, indeed. Interesting, but it does show in the Mn distributions.
L521 Sulfate reduction is very important bit only in the marine realm where sulfate is 28 mM. Here we have very low levels in the order of 100 µM. That is even for rivers very low!
L644 the conceptual model is 1D, right? In the discussion and the model lateral transport is entirely missing. Please check with literature from the waddensea, e.g. by Engelen, Cypionka and Beck on deep biosphere activities in the shallow intertidal flats. See also the review from Joye on transport phenomena and the activities at 20 m depth (doi.org/10.1016/B978-0-444-63893-9.00012-5).
Citation: https://doi.org/10.5194/bg-2023-62-RC1 -
AC1: 'Reply on RC1', Duteil Thibault, 30 Jun 2023
Dear editor,
On behalf of my co-authors, I am submitting a revised version of our manuscript 2023-62 to be considered for publication in Biogeosciences. We have addressed the reviewer’s concerns and questions, either by adding complementary data/observations and/or by justifying our original statements. As requested by reviewer #1, we incorporate the advective transport phenomenon throughout the manuscript, which greatly improved the introduction and discussion sections. We also included additional information about sulfate reduction and methanogenesis, as requested by reviewer #2. Overall, the comments were much appreciated and helped to improve the manuscript. We look forward to your feedback and decision.
Best regards,
Thibault Duteil
General remark to editor and reviewers:
- Four figures (Figures 1, 2, 3 and 10) have been slightly modified compared to the original manuscript and a new figure has been added (Figure 9).
- Parts of the text that were modified or displaced in the original manuscript are written in red.
- Several references recommended by the reviewers or which refine the discussion have been added to the original manuscript.
-Response to Reviewer #1, Dirk De Beer:
The paper describes a biogeochemical study towards activities in deep estuarine sediments. A broad suite of methods is used, mostly in a correct way. Concluded is that also deep in sediments, below several meters significant activities occur. The discussion lacks depth, not all strange results are recognized, sedimentation time is not considered, nor transport phenomena.
-The paper is written in good English, but poor style, making it hard to read. The text in the result section consists of summation of numbers of the plots and table. The text should rather describe the figures and point to the remarkable features, give a first interpretation, that later may be discussed. It is exceptionally difficult reading and not informative. The figures require some basic interpretation for readability, that is missing. The authors should tell us a story, not provide a list of dry facts. Unfortunately, this section must be completely rewritten to make a fluent and readable text
- We understand that this part may require careful reading, but as it contains a large dataset comprising many geochemical, sedimentological and microbial measurements, we feel strongly that this section needs to have a certain level of detail. We prefer to keep most of the original observations (Results) separate from interpretations (Discussion), making results of our study as transparent and clear as possible. However, we took the reviewer’s comment into account and (i) simplified and shortened the Results section in which (ii) we rewrote the part on microbial activity. We trust that this clarifies the results section and improves the readability of the paper.
-The summary is good and invites to further reading. The introduction is acceptable but a bit short, focusing on the well-known redox cascade. However, the essential introduction to transport processes in coastal sediments is missing. The rational for the sample location should be described. The location is not ideal, with many complicating influences. The methods is detailed, can be improved at some minor points. The result section is described above. Also in the discussion deep advection, that is often essential for understanding of deep biosphere activities and certainly for close to coast permeable sediments, is missing.
- We agree with the reviewer’s comments. Consequently, we will expand the introduction and discussion to include transport and lateral advection phenomena, notably to explain the nitrate peak at the surface and the concomitant sulfate and nitrate peaks present at depth. Furthermore, we included two recent references on the topic of transport (Ahmerkamp et al., 2017; Schutte et al. 2019) to improve the discussion of this process. Following the reviewer’s recommendation, the text has been modified in the introduction and the discussion part. We appreciate the reviewer’s comment as it improves the discussion section. We have also specified sample location (Figure 1). The justification for the location of the cores is developed in answer n°3 (see below).
Ahmerkamp, S., Winter, C., Krämer, K., Beer, D. de, Janssen, F., Friedrich, J., Kuypers, M. M. M., and Holtappels, M.: Regulation of benthic oxygen fluxes in permeable sediments of the coastal ocean: Regulation of benthic oxygen fluxes, Limnol. Oceanogr., 62, 1935–1954, https://doi.org/10.1002/lno.10544, 2017.
Schutte, C. A., Ahmerkamp, S., Wu, C. S., Seidel, M., de Beer, D., Cook, P. L. M., and Joye, S. B.: Chapter 12 - Biogeochemical Dynamics of Coastal Tidal Flats, in: Coastal Wetlands (Second Edition), edited by: Perillo, G. M. E., Wolanski, E., Cahoon, D. R., and Hopkinson, C. S., Elsevier, 407–440, https://doi.org/10.1016/B978-0-444-63893-9.00012-5, 2019.
Understanding of transport phenomena, especially in such ‘difficult’ sites is crucial. The site is very difficult, as it is a freshwater site with some marine influence, in a flowing river and very likely with lateral groundwater input. On top of that, the location is in a river bend, with irregular sedimentation.
- We agree with the reviewer’s comment that our site is subject to various surface and underground transport processes and thus complex. The Bordeaux North point bar was selected based on results from previous investigations and remains relatively undisturbed compared to other locations in the Gironde Estuary (e.g., no dredging activity). There is tidal influence present at the site, yet the salinity remains low (between 0.2 and 5 ppt; MAGEST; https://magest.oasu.u-bordeaux.fr/). It is indeed a complex environment where coring is challenging, but we previously drilled three long cores (4-6 meter long), which were studied for sedimentology and petrography (e.g., Virolle et al., 2021). These previous cores allowed to better constrain the point stratigraphic architecture and locate the coring area for this study. Finally, the Bordeaux North point bar is subject to significant sediment deposition and preservation (average sedimentation rate of more than 3 cm.yr-1) since its initiation ca. 300 yrs ago (Virolle et al., 2021). All these reasons justified the choice of this point bar for our study. The hydro-sedimentary context of the study area is now explained in a new section “Hydrological and Sedimentological context” just following the introduction. We think this clarifies the context of the study. Furthermore, the Bordeaux harbor bathymetric maps allowed us to build a cross section linking the sedimentology of the core with the point bar internal stratigraphic architecture (Figure 1; see attached document). The revised version of the paper will include a new sub-section describing this internal architecture and the point bar chronostratigraphy in the results section. We will also add a new figure where geochemical depth profiles and sediment porosity of the Long Core are plotted on this cross section (Figure 9; see attached document). This figure allowed us to support the hypothesis of lateral advection in the permeable horizons of the point bar as suggested by the reviewer, and greatly enhanced the discussion.
MAGEST: https://magest.oasu.u-bordeaux.fr/
Virolle, M., Brigaud, B., Féniès, H., Bourillot, R., Portier, E., Patrier, P., Derriennic, H., and Beaufort, D.: Preservation and distribution of detrital clay coats in a modern estuarine heterolithic point bar in the Gironde estuary (Bordeaux, France), Journal of Sedimentary Research, 91, 812–832, https://doi.org/10.2110/jsr.2020.146, 2021.
The figures are made with care, but on several places the letters and numbers must be made larger.
- We increased the size of the font where it was too small. We think the figures are now more readable thanks for pointing that out.
Some details:
-From Figure 1 we cannot understand where the 3 cores are taken. Firstly, the 2 maps in 1A should be aligned so that North is on top. Indicated is the location of the box and short core, not of the long core. It is probably not the same spot as the geochemistries are different. The drawings are nice, but the text should be much larger. The depth axes labels are invisibly tiny and of poor quality. I recommend to make more space for the plots.
- In the literature, the Gironde estuary is commonly represented as shown in Figure 1 (e.g., Virolle et al., 2021). The position of the long core has been added in the figure as requested and the text in the figure, including that for depth axes, has been enlarged (Figure 1, see attached document).
Virolle, M., Brigaud, B., Féniès, H., Bourillot, R., Portier, E., Patrier, P., Derriennic, H., and Beaufort, D.: Preservation and distribution of detrital clay coats in a modern estuarine heterolithic point bar in the Gironde estuary (Bordeaux, France), Journal of Sedimentary Research, 91, 812–832, https://doi.org/10.2110/jsr.2020.146, 2021.
Explain what is a mud pebble and a mud drape.
- A mud pebble is a centimeter sized rounded particle composed of mud (i.e., a mix of clay and silt-sized particles). Mud pebbles mostly derive from the erosion of low energy intertidal sediments by tidal currents. A mud drape is a centimeter-thick layer of mud deposited at the surface of the dunes and in inter-dune depressions during periods of high and low tide slack water. Both definitions have been added to the Core sedimentary description, sub-section of the Results.
What is the meaning of the letters at the bottom right?
- The bottom letters correspond to the grain size in the sedimentary sections. This is now clarified in the legend of Figures 1 and 2.
-All cores have the label BXN, better remove that as it does not help keeping them apart.
- We agree with the reviewer’s comment and removed the label BXN.
-How are the blanks, so the killed controls, treated for the metabolic assays by TTC and FDA?
- Blanks were prepared by adding 2 mL of 1.5% glutaraldehyde to the sediment and treated in the same manner as live samples. We have added two sentences in the method part to clarify this.
-In Fig 2 the letters and numbers are far too small. Instead of repeating these plots in numbers in the text, describe what we see and what is remarkable. What I find remarkable is the multiple peaks in the sulfate, Fe and Mn plots. Very strange is the high nitrate peak in the sulfidic zone. These observations are important as it is impossible in a 1D interpretation: they can only be result of lateral transport through permeable layers. Thus best is to align these plots with porosity plot or at least the geological info in Fig 1 (mud, sand etc).
- We agree with the reviewer’s comment and added a figure where we align porewater composition and sediment porosity (as measured on thin sections) with the 2D cross section of the point bar (figure 9; see attached document). This figure confirms potential lateral advection of sulfates and nitrates through permeable sand layers, as suggested by the reviewer. This would explain the peaks measured at depth. See answer n°12 below for a detailed response about the nitrate peaks and origin. The discussion has also been expanded, in particular to include potential lateral transport of these solutes through permeable sandy horizons.
-In Fig 3 we see again peaks, and nitrate and sulfate correlate: lateral input. Fe2+ is absent where nitrate peaks, nice. The FeIII plots shows impossible concentrations, the method cannot be correct. FeIII does not dissolve well, certainly not to µM levels.
- We occasionally observed a red precipitate in the Long Core pore water. It is conceivable that there was iron exchange between the colloids and the water, causing this red precipitation. Jones et al. (2011) reported a similar observation in the Satilla River estuary. These authors hypothesized that soluble Fe(III) complexes could be released into the pore waters through reequilibration with the surrounding sediment. We agree that the Fe(III) concentration seems high, however, not impossible. Our values are in the same order of magnitude as reported by e.g., Hopwood et al., 2014, or Beckler et al., 2015 in estuarine porewater. An alternative explanation could be a long term equilibration between porewater and previously precipitated minerals susceptible to release iron, such as iron sulfides. Such minerals would precipitate in the sulfate reduction zone and subsequently dissolve due to slightly more oxidizing conditions deeper in the sediment. Audry et al. (2006) estimated that up to 0.8 mM of iron could be released in the Gironde mud upon iron sulfide dissolution. Beck et al. (2008) also reported concentrations of up to 500 µM of dissolved Fe 5 meters deep in the Wadden Sea intertidal flats. In the revised version of the paper, we will also add measurements of total organic carbon (TOC) in the Long Core sediment (Figure 3; see attached document). We observed a low, but positive correlation between dissolved Fe and TOC, which could indicate the presence of colloidal DOM-Fe complexes in porewater. These different possibilities will be included in the discussion for more clarity. We are grateful for the reviewer’s suggestions to consult the literature on the Wadden Sea which enhanced the discussion.
Audry, S., Blanc, G., Schäfer, J., Chaillou, G., and Robert, S.: Early diagenesis of trace metals (Cd, Cu, Co, Ni, U, Mo, and V) in the freshwater reaches of a macrotidal estuary, Geochimica et Cosmochimica Acta, 70, 2264–2282, https://doi.org/10.1016/j.gca.2006.02.001, 2006.
Beck, M., Dellwig, O., Schnetger, B., and Brumsack, H.-J.: Cycling of trace metals (Mn, Fe, Mo, U, V, Cr) in deep pore waters of intertidal flat sediments, Geochimica et Cosmochimica Acta, 72, 2822–2840, https://doi.org/10.1016/j.gca.2008.04.013, 2008.
Beckler, J. S., Jones, M. E., and Taillefert, M.: The origin, composition, and reactivity of dissolved iron (III) complexes in coastal organic-and iron-rich sediments, Geochimica et Cosmochimica Acta, 152, 72–88, https://doi.org/10.1016/j.gca.2014.12.017, 2015.
Hopwood, M. J., Statham, P. J., and Milani, A.: Dissolved Fe (II) in a river-estuary system rich in dissolved organic matter, Estuarine, Coastal and Shelf Science, 151, 1–9, https://doi.org/10.1016/j.ecss.2014.09.015, 2014.
Jones, M. E., Beckler, J. S., and Taillefert, M.: The flux of soluble organic‐iron (III) complexes from sediments represents a source of stable iron (III) to estuarine waters and to the continental shelf, Limnology and Oceanography, 56, 1811–1823, https://doi.org/10.4319/lo.2011.56.5.1811, 2011.
Very strange is to have high nitrate levels down to 1.3 m depth. Must be advection and low conversion. Is the land next to the river cultivated and fertilized intensively?
- The land abutting the site is an industrial zone. Further upstream as well as downstream from the sampling site are vineyards and land exposed to other agricultural activities. As the reviewer suggests, it is plausible that there are terrestrial inputs from the above-mentioned sources of various nutrients, notably nitrogen and phosphorus (the latter was not measured). We now included in the discussion on lateral flow or, in other words, advective transport (see above) that this could have affected the porewater depth profile of nitrate, and possibly sulfate. It should be noted that the nitrate peak is present in a coarse and permeable sand horizon, corroborating the lateral transport hypothesis (Figure 9; see attached document). The lateral transport (i.e, nitrate flux) can be further modulated by tidal pumping.
-The concentrations of sulfate and Fe differ strongly between the long and short cores. Why?
- Sediment properties vary greatly by location. Even within close proximity there can be a high degree of (lateral) variability, as much as two orders of magnitude, as shown by Charette & Sholkovitz (2006) and Rouxel et al. (2008) in Waquoit Bay (USA) or El Ghobary (1982) and Audry et al. (2006) in the Gironde Estuary. This point has been added to the discussion. Clay layers, as present in Long Core, can be a source for Fe, which could further explain the discrepancy between the Fe values in the two cores.
Audry, S., Blanc, G., Schäfer, J., Chaillou, G., and Robert, S.: Early diagenesis of trace metals (Cd, Cu, Co, Ni, U, Mo, and V) in the freshwater reaches of a macrotidal estuary, Geochimica et Cosmochimica Acta, 70, 2264–2282, https://doi.org/10.1016/j.gca.2006.02.001, 2006.
Charette, M. A. and Sholkovitz, E. R.: Trace element cycling in a subterranean estuary: Part 2. Geochemistry of the pore water, Geochimica et Cosmochimica Acta, 70, 811–826, https://doi.org/10.1016/j.gca.2005.10.019, 2006.
El Ghobary, H.: Interstitial water chemistry of the inner continental shelf sediments off the Gironde Estuary, Estuarine, Coastal and Shelf Science, 16, 639–650, https://doi.org/10.1016/0272-7714(83)90076-8, 1983.
Rouxel, O., Sholkovitz, E., Charette, M., and Edwards, K. J.: Iron isotope fractionation in subterranean estuaries, Geochimica et Cosmochimica Acta, 72, 3413–3430, https://doi.org/10.1016/j.gca.2008.05.001, 2008.
-The paragraph on microbial activities has almost more numbers than text. Unreadable. The hydrolytic activity seems to be hardly decreasing with depth, that is possible, but unlikely. Did you correct for killed controls? Same for the TCC release. Both do not relate with the peak in metabolic activity measured by calorimetry (Table 1). What do you actually measure with TCC release?
- The controls for both TTC and FDA showed zero activity and thus corrections were not necessary. It is common to have different results from these assays and calorimetry. Indeed, although these assays have the same purpose (i.e., measuring metabolic activity), they have rather different targets. The TTC assay is a direct measurement of metabolic activity through reduction of tetrazolium by reductases mostly from the electron transport system (ETS). The FDA assay measures hydrolytic activity of ubiquitous lipases, proteases, and esterases enzymes (i.e., non-specific hydrolases and their capacity to degrade large molecules). We assume that FDA remains high because some of these enzymes (proteases for example) are often quite resistant themselves to degradation once produced; they will continue hydrolyzing their substrate even if the microbial activity has decreased or stopped (Mayer, 1989). While hydrolytic activity (FDA) is positively correlated to TTC (r=0.55; p<0.05; Figure 7), but it shows a strong anticorrelation to porewater pH (r=-0.7; p<0.05; Figure 7). A more acidic pH (ca. 7.9; 0-5 cm) is associated with a more elevated hydrolytic activity, while a higher pH (ca. 8.1-8.2; below 5 cm) is associated with a lower FDA hydrolysis. This point will be added to the discussion of the revised manuscript. The calorimetric assays measure the capacity of the metabolic heat resulting from the activity of microbial community. Isothermal calorimetry contrary to TTC and FDA is not an end-point assay but measures the heatflow in real time, thus providing insight on the time for the microbial community to adapt to the substrate (the lag phase) see next point as well (15). These three assays measure different types of activities but are complementary to each other. They might not yield similar results but show that there are active microbial communities present at different depths albeit with different properties. We will try to make this as clear as possible in the revised version of the manuscript. See detailed review of these assays in Braissant et al., 2020.
Braissant, O., Astasov-Frauenhoffer, M., Waltimo, T., and Bonkat, G.: A review of methods to determine viability, vitality, and metabolic rates in microbiology, Frontiers in Microbiology, 11, 547458, https://doi.org/10.3389/fmicb.2020.547458, 2020.
Mayer, L. M.: Extracellular proteolytic enzyme activity in sediments of an intertidal mudflat, Limnology and Oceanography, 34, 973–981, https://doi.org/10.4319/lo.1989.34.6.0973, 1989.
-The metabolic activity peaks at 50 cm depth, according to table 1. Explain what drives this high rate.
- We will add the value of the surface biofilm to clarify table 1. Generally, the activity tends to decrease with sediment depth, as indicated by the maximum heatflow. There is an even clearer decrease in the heat released Q with depth suggesting that deeper sediments are less prone to support growth (heat being considered as a proxy for the biomass formed or substrate consumed). The sample taken at 50 cm stands out of this overall trend. Indeed, although the microbial community present at 50 cm depth showed a lower heat production compared to surface, it also showed a high activity as well as a rather extended lag phase before consumption of the amended substrate (the higher activity is also supported by the TTC assays that is higher at 50 cm compared to the neighboring samples in the core; Figure 4). This means that the presence of fresh substrate invokes a gene expression in the microbial community that is followed by de novo protein (i.e., enzyme) synthesis. Alternatively, the addition of fresh substrates might trigger spore germination (also relying on the processes described before). Once substrate consumption starts, the activity and the growth rate are indeed higher at 50 cm than in the three other layers. However, the population size at this depth is an order of magnitude smaller than in the other layers. Our CFU counts in this case might not take into account a large proportion of spores from so called strict anaerobes (especially from sulfate reducing bacteria). This is a limitation of our study that must be recognized. In this context, we hypothesize that the germination of spores (from aerobes and anaerobes) as well as the time required for de novo protein (i.e., enzyme) synthesis are responsible for the longer lag time observed (an additional 3 hours compared to all other samples; spore germination is in the order of 150 minutes see ref below). The combined germination of spores in addition to the community present in the sample might result in the high growth rate and activity observed because of the high number of microbes suddenly “reactivated” and growing at the same time. Such hypothesis fits well with the often large proportion of spores observed in sediments (both aerobes and anaerobes; Jorgensen, 2012) and with the time required for a spore to become a growing vegetative cell again (around 150 minutes - see refs below). Also, several spore forming bacteria are known to grow rather fast. Members of the genus Bacillus are good examples, as well as Clostridiales. The origin of those spores cannot be attributed to endogenous or exogenous origin, our observations and the current literature suggest that they could play an important role when substrates become available.
Cupit, C., Lomstein, B. A., and Kjeldsen, K. U.: Contrasting community composition of endospores and vegetative Firmicutes in a marine sediment suggests both endogenous and exogenous sources of endospore accumulation, Environmental Microbiology Reports, 11, 352–360, https://doi.org/10.1111/1758-2229.12679, 2019.
Jørgensen, B. B.: Shrinking majority of the deep biosphere, Proceedings of the National Academy of Sciences, 109, 15976–15977, https://doi.org/10.1073/pnas.12136391, 2012.
O’Sullivan, L. A., Roussel, E. G., Weightman, A. J., Webster, G., Hubert, C. R., Bell, E., Head, I., Sass, H., and Parkes, R. J.: Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure, ISME J, 9, 922–933, https://doi.org/10.1038/ismej.2014.190, 2015.
Sinai, L., Rosenberg, A., Smith, Y., Segev, E., and Ben-Yehuda, S.: The Molecular Timeline of a Reviving Bacterial Spore, Molecular Cell, 57, 695–707, https://doi.org/10.1016/j.molcel.2014.12.019, 2015.
-Also the description of the XRF data is sedative. The plots show that the distributions of metals and S correlate. Compare again with the sedimentary log of Fig 1.
- The distribution of solid metals (Fe, Mn, Al, K) as measured with XRF is largely influenced by the mineralogical nature of the sediments. Clay-rich sediments have relatively high abundance of metals, whereas sands are metal-poor. While we agree that sulfur is correlated with metals in the Box and Short Core, this is not the case in the Long Core. We interpret the parallel increase in sulfur and metal concentrations (as observed in the Box and Short Core between 15 and 40 centimeters) to reflect precipitation of metal-sulfides or clay (Al-S) in the sulfate reduction zone. In Long Core, sulfur and metals sometimes correlate well, and if not, S decrease appears to occur in oxidized zones (especially at greater depth) as indicated by the reddish color of oxidized iron (Fe(III)). Such an oxidized horizon can be observed at 3.5 meters in the long core, where a reddish colors correlates with a decrease of sulfur in XRF (Figure S4).
-The discussion lacks depth. It is a summary of possible microbial conversions. Transport is missing.
- We agree and will add a sub-section on underground water transport (e.g., advection process) in the discussion. As suggested by the reviewer, its potential role on lateral transport of e.g., nitrates in the sediment is now discussed, greatly improving the discussion.
-L 504 accumulation of nitrate by sulfide is nonsense. Many bacteria oxidise sulfide by nitrate.
- We agree with the reviewer that in the absence of oxygen (or under very low oxygen concentrations) sulfide is oxidized with nitrate. However, there are several reports in the literature that even very low sulfide concentrations can inhibit denitrification, notably N2O reduction, in laboratory (Sorensen et al., 1980; Delgado Vela et al., 2018), in brackish and fresh water systems (Aelion & Warttinger, 2010) and in estuaries (Senga et al., 2005). Sulfide may also impact other metabolisms linked with nitrate as for example inhibits Annamox (Carvajal-Arroyo et al., 2013; Jin et al., 2013; Wisniewski et al., 2019). We prefer to include this possibility, but modified the text to indicate that this is a less likely scenario in the discussion part.
Aelion, C. M. and Warttinger, U.: Sulfide inhibition of nitrate removal in coastal sediments, Estuaries and Coasts, 33, 798–803, https://doi.org/10.1007/s12237-010-9275-4, 2010.
Carvajal-Arroyo, J. M., Sun, W., Sierra-Alvarez, R., and Field, J. A.: Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents, Chemosphere, 91, 22–27, https://doi.org/10.1016/j.chemosphere.2012.11.025, 2013.
Delgado Vela, J., Dick, G. J., and Love, N. G.: Sulfide inhibition of nitrite oxidation in activated sludge depends on microbial community composition, Water Research, 138, 241–249, https://doi.org/10.1016/j.watres.2018.03.047, 2018.
Jin, R.-C., Yang, G.-F., Zhang, Q.-Q., Ma, C., Yu, J.-J., and Xing, B.-S.: The effect of sulfide inhibition on the ANAMMOX process, Water Research, 47, 1459–1469, https://doi.org/10.1016/j.watres.2012.12.018, 2013.
Senga, Y., Mochida, K., Fukumori, R., Okamoto, N., and Seike, Y.: N2O accumulation in estuarine and coastal sediments: the influence of H2S on dissimilatory nitrate reduction, Estuarine, Coastal and Shelf Science, 67, 231–238, https://doi.org/10.1016/j.ecss.2005.11.021, 2006.
Sørensen, J., Tiedje, J. M., and Firestone, R. B.: Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens, Applied and Environmental Microbiology, 39, 105–108, https://doi.org/10.1128/aem.39.1.105-108.1980, 1980.
Wisniewski, K., Di Biase, A., Munz, G., Oleszkiewicz, J. A., and Makinia, J.: Kinetic characterization of hydrogen sulfide inhibition of suspended anammox biomass from a membrane bioreactor, Biochemical Engineering Journal, 143, 48–57, https://doi.org/10.1016/j.bej.2018.12.015, 2019.
-L 509 MnO2 driving ammonium oxidation has been mentioned before, indeed. Interesting, but it does show in the Mn distributions.
- The nitrate peak area coincides with an increase in dissolved manganese concentration. Not knowing which hypothesis is the correct one to explain the nitrate peak, we would like to mention all possible scenarios including this one.
-L521 Sulfate reduction is very important bit only in the marine realm where sulfate is 28 mM. Here we have very low levels in the order of 100 µM. That is even for rivers very low!
- As the reviewer points out, the concentration of sulfate is indeed very low (10-100 µM). We report measurements of sulfide in our study, thus reasonably assuming the presence of sulfate reduction (Cypionka et al., 1985; Widdel et al., 1992). Holmkvist et al. (2011) showed sulfate reducing activity below the sulfate-methane transition zone in a nearshore marine sediment despite negligible sulfate concentrations. A recent study by Marietou et al. (2021) reported a km (half saturation constant) for sulfate of 4 µM for a marine sulfate reducing microbe. Based on this, we believe that sulfate reduction can be invoked as one of the microbial metabolisms in our study.
Cypionka, H., Widdel, F., and Pfennig, N.: Survival of sulfate-reducing bacteria after oxygen stress, and growth in sulfate-free oxygen-sulfide gradients, FEMS Microbiology Letters, 31, 39–45, https://doi.org/10.1111/j.1574-6968.1985.tb01129.x, 1985.
Holmkvist, L., Ferdelman, T. G., and Jørgensen, B. B.: A cryptic sulfur cycle driven by iron in the methane zone of marine sediment (Aarhus Bay, Denmark), Geochimica et Cosmochimica Acta, 75, 3581–3599, https://doi.org/10.1016/j.gca.2011.03.033, 2011.
Marietou, A., Kjeldsen, K. U., Glombitza, C., and Jørgensen, B. B.: Response to substrate limitation by a marine sulfate-reducing bacterium, ISME J, 16, 200–210, https://doi.org/10.1038/s41396-021-01061-2, 2022.
Widdel, F. and Bak, F.: Gram-Negative Mesophilic Sulfate-Reducing Bacteria, in: The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, edited by: Balows, A., Trüper, H. G., Dworkin, M., Harder, W., and Schleifer, K.-H., Springer, New York, NY, 3352–3378, https://doi.org/10.1007/978-1-4757-2191-1_21, 1992.
-L644 the conceptual model is 1D, right? In the discussion and the model lateral transport is entirely missing. Please check with literature from the waddensea, e.g. by Engelen, Cypionka and Beck on deep biosphere activities in the shallow intertidal flats. See also the review from Joye on transport phenomena and the activities at 20 m depth (doi.org/10.1016/B978-0-444-63893-9.00012-5).
21. Good remark, we added lateral transport in the model as another manner to explain geochemical gradients measured in this study (notably nitrate and sulfate peaks; Figure 10). In order to improve the model, we will add a 2D sketch at the point bar scale in the model (Fig. 10C). This larger scale sketch will help linking sedimentological and biogeochemical data and to discuss the role of lateral advection. The text describing the model has been updated as well in the discussion part. We would like to thank the reviewer for this relevant comment.
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AC1: 'Reply on RC1', Duteil Thibault, 30 Jun 2023
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RC2: 'Comment on bg-2023-62', Anonymous Referee #2, 15 May 2023
Duteil et al. conducted a series of geochemical and microbiological analyses on sediment cores from the Garonne estuarine channel. Based on porewater and solid-phase geochemical profiles, the authors define the biogeochemical zonation of the sediment cores. Good spatial resolution is achieved in the upper 25 cm, whereas low-resolution profiles of deeper sediments hinder the discussion on certain processes. Microbial activity data are used to facilitate the discussion on biogeochemical processes, but they are not specific to certain processes, which limits their further application. Nevertheless, I found some of the conclusions are justified by the observation and the utilization of metabolic assays and isothermal calorimetry are novel in this type of work. I have several concerns about the interpretation of porewater geochemical profiles as detailed below.
Line 35-37. Given the low resolution of geochemical profiles in the subsurface, I would suggest avoiding defining certain biogeochemical processes to occur at exact depth intervals. For example, it seems to me that iron reduction occurs below 16 cm and manganese reduction occurs below 2 cm to the lower part of the short core. In addition, metal reduction can well be due to abiotic processes involving sulfide oxidation, as the authors mentioned later in the discussion. Therefore, I would suggest interpreting their significance more carefully when discussing their presence in the context of microbial metabolism.
Line 25. “Restricted to the upper decimeters of the sediment column” and “rarely from direct measurements of microbial activity” are not accurate. Porewater geochemical profiles and rates of sulfate reduction and methanogenesis have been frequently measured in both surface and subsurface marine sediments.
Line 96-97. “Potential links between microbial metabolisms and early diagenesis” has been widely studied not only to depths of around 20 cm but also in meter- or kilometer-long sediment cores.
Line 148. The methodology for the analysis of sulfate concentrations and its detection limit is not described. In Figure 3B, the sulfate concentrations are all lower than 12 μM, which is often the detection limit of ion chromatography.
Line 535. Given the extremely low concentrations of sulfate in the subsurface and the low resolution of the sulfate profile below 25 cm, I am not convinced that sulfate reduction occurs below 25 cm in the short core or throughout the long core. My concern is supported by the sulfide profile, in which the sulfide concentrations only increase with depth in the upper 20 cm.
Line 572. Following the comment above, I would instead suggest that methanogenesis occurs from shallow depth (e.g., 25 cm or 1 m) to the deep subsurface. Discrete methanogenic zones are not commonly observed in natural environments.
Citation: https://doi.org/10.5194/bg-2023-62-RC2 -
AC2: 'Reply on RC2', Duteil Thibault, 30 Jun 2023
Dear editor,
On behalf of my co-authors, I am submitting a revised version of our manuscript 2023-62 to be considered for publication in Biogeosciences. We have addressed the reviewer’s concerns and questions, either by adding complementary data/observations and/or by justifying our original statements. As requested by reviewer #1, we incorporate the advective transport phenomenon throughout the manuscript, which greatly improved the introduction and discussion sections. We also included additional information about sulfate reduction and methanogenesis, as requested by reviewer #2. Overall, the comments were much appreciated and helped to improve the manuscript. We look forward to your feedback and decision.
Best regards,
Thibault Duteil
General remark to editor and reviewers:
- Four figures (Figures 1, 2, 3 and 10) have been slightly modified compared to the original manuscript and a new figure has been added (Figure 9).
- Parts of the text that were modified or displaced in the original manuscript are written in red.
- Several references recommended by the reviewers or which refine the discussion have been added to the original manuscript.
Second reviewer:
Duteil et al. conducted a series of geochemical and microbiological analyses on sediment cores from the Garonne estuarine channel. Based on porewater and solid-phase geochemical profiles, the authors define the biogeochemical zonation of the sediment cores. Good spatial resolution is achieved in the upper 25 cm, whereas low-resolution profiles of deeper sediments hinder the discussion on certain processes. Microbial activity data are used to facilitate the discussion on biogeochemical processes, but they are not specific to certain processes, which limits their further application. Nevertheless, I found some of the conclusions are justified by the observation and the utilization of metabolic assays and isothermal calorimetry are novel in this type of work. I have several concerns about the interpretation of porewater geochemical profiles as detailed below.
-Line 35-37. Given the low resolution of geochemical profiles in the subsurface, I would suggest avoiding defining certain biogeochemical processes to occur at exact depth intervals. For example, it seems to me that iron reduction occurs below 16 cm and manganese reduction occurs below 2 cm to the lower part of the short core. In addition, metal reduction can well be due to abiotic processes involving sulfide oxidation, as the authors mentioned later in the discussion. Therefore, I would suggest interpreting their significance more carefully when discussing their presence in the context of microbial metabolism.
22. We appreciate the reviewer’s recommendation. Following his suggestion, we changed the sentence and made more general statements about specific processes without referring to exact depth intervals in the abstract and the discussion parts. We will also be more temperate in the discussion about the conceptual biogeochemical model.
-Line 25. “Restricted to the upper decimeters of the sediment column” and “rarely from direct measurements of microbial activity” are not accurate. Porewater geochemical profiles and rates of sulfate reduction and methanogenesis have been frequently measured in both surface and subsurface marine sediments.
23. Here, we intended to discuss direct measurements of microbial activity, not the rate of metabolic reactions. However, we agree with the reviewer that this sentence is easily misinterpreted. In order to avoid any misunderstanding, we removed the part of lacking microbial measurements from this sentence.
-Line 96-97. “Potential links between microbial metabolisms and early diagenesis” has been widely studied not only to depths of around 20 cm but also in meter- or kilometer-long sediment cores.
24. Per the reviewer’s recommendation, we changed this sentence.
-Line 148. The methodology for the analysis of sulfate concentrations and its detection limit is not described. In Figure 3B, the sulfate concentrations are all lower than 12 μM, which is often the detection limit of ion chromatography.
25. We agree with the reviewer’s comment. The analysis of sulfate should have been included with assay of the other ions, using the ion chromatography. This was corrected in the method section. The detection limit of suppressed ion chromatography is around 0.1 uM, which is lower than the reviewer indicated in this comment. However, we agree with the reviewer that such low concentrations make interpretation of sulfate/sulfide/sulfur cycling complicated and thus we have been more careful when discussing the long core sulfate observations.
-Line 535. Given the extremely low concentrations of sulfate in the subsurface and the low resolution of the sulfate profile below 25 cm, I am not convinced that sulfate reduction occurs below 25 cm in the short core or throughout the long core. My concern is supported by the sulfide profile, in which the sulfide concentrations only increase with depth in the upper 20 cm.
26. We agree with the reviewer that the low spatial resolution associated with analyses of the long core makes it more challenging to identify specific trends in certain metabolisms. The localization of the sulfate reduction in the model (Figure 10) has also been changed. The low sulfate concentrations and decrease with depth (Figures 2 and 3) could, however, be indicative of sulfate reduction. As we mention in the text, in addition to possibly minor reoxidation of sulfide to intermediates other than sulfate (zero-valent sulfur, thiosulfate, polythionates, polysulfides; Visscher & Van Gemerden 1993; Megonigal et al. 2003), sulfides could be associated with metals such as iron (refer to the X-ray fluorescence profiles, Figures 6 and S4). Furthermore (now discussed in much greater detail per Reviewer #1 recommendation), advective (lateral) transport through permeable sandy layers may further distort depth profiles, e.g., locally reoxidizing the sediment, or supplying sulfate or nitrate. For this reason, we carefully described the stratigraphy/architecture by adding a specific sub-section in the results (Point bar architecture and chronostratigraphy) of the sediment column in each of the cores we obtained in order to interpret with care the sulfide data. For further explanations, see our rebuttal on the comment of reviewer 1 (answer 20, about L521) on this topic.
Megonigal, J. P., Hines, M. E., and Visscher, P. T.: Anaerobic metabolism: linkages to trace gases and aerobic processes, Biogeochemistry, 8, 317–424, https://doi.org/10.1016/B0-08-043751-6/08132-9, 2003.
Visscher, P. T. and Van Gemerden: Sulfur Cycling in Laminated Marine Microbial Ecosystems | SpringerLink, 1993.
-Line 572. Following the comment above, I would instead suggest that methanogenesis occurs from shallow depth (e.g., 25 cm or 1 m) to the deep subsurface. Discrete methanogenic zones are not commonly observed in natural environments.
27. We agree with the reviewer that typically, methanogenesis below the sulfate-methane transition zone is continuous and methane concentrations (not measured in our study), should increase with depth (Megonigal et al. 2003). However, as shown in Figure 3, for several reasons (e.g., rapid burial of sediments, lateral advection in coarse sand layers) the organic carbon distribution is heterogeneous. It is therefore conceivable, with some care given the larger intervals of which we made observations, that there are certain sedimentary horizons associated with higher carbon contents that support higher rates of methanogenesis. To support this hypothesis, we have added the concentrations of total organic carbon (TOC) in the long core (Figure 3; see attached document). We will also modify the discussion to include TOC data for a more in-depth discussion on this aspect.
Megonigal, J. P., Hines, M. E., and Visscher, P. T.: Anaerobic metabolism: linkages to trace gases and aerobic processes, Biogeochemistry, 8, 317–424, https://doi.org/10.1016/B0-08-043751-6/08132-9, 2003.
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