| 25 Aug 2026
Biogeochemical and microbial microseepage connectivity from a deeply buried salt diapir on the Scotian Slope of Atlantic Canada
Abstract. Cold seeps are seafloor environments where hydrocarbon-rich fluids, including methane and hydrogen sulfide, migrate through deep-seated faults and fractures to the seabed, supporting chemosynthetic communities. Although the biological activity within and immediately around these sites can be highly elevated compared to the surrounding deep-ocean ambient sediments, little is known about whether subtle changes extend to the larger periphery, where no visible signs of seepage exist. Here we examine how geological structures, such as subsurface faults, regulate seep formation and fluid migration, and assess the influence this has on microbial distributions by combining a 2.5×2.5×1 km three-dimensional seismic survey with a 1.6 km transect study that overlies a deeply buried salt diapir. The crest of the diapir rests ~1 km below the seafloor, where it has heavily disturbed the overlying bedrock with radial and crustal faults, half-graben block displacements, and salt-tectonic-influenced anticlinal mini-basins. Rocks draping the diapir also host a subsurface methanogenic deep biosphere that produces sufficient methane to sustain a biodiverse ocean floor cold seep. A portion of the produced hydrocarbons has become trapped within ~300 mbsf block-faulted strata where they are recognizable in seismic data as a direct hydrocarbon indicator (DHI). We analyzed 52 sediment samples extending 5–575 cm below seafloor (cmbsf) from five gravity cores and one push core collected at an active cold seep. Spatial heterogeneity in cored sediments along the transect was revealed by porewater geochemistry, stable isotopes, and lipidomics. Collectively, these data resolve distinct chemosynthetic zones that form tightly coupled functional networks mediated by interlinked redox-dependent cycling of nitrogen, carbon, iron, and manganese that are comprised of ammonia-oxidizing archaea (AOA), anaerobic methanotrophs (ANMEs), and sulfate-reducing bacteria (SRB). Evidence is provided that microbial assemblages and their metabolic interactions are shaped and sustained by diffuse and largely non-detectable microseepage ascending along more deeply buried fault systems associated with the salt diapir. The results, therefore, establish a spatially resolved model for offshore geochemical surveys that link subsurface fluid flow to redox gradients and microbial activity.
Summary
In this manuscript Oueslati and co-authors present biogeochemical and biomarker data from a range of short gravity/push cores that were taken along a transect across a deeply buried salt diapir in the Atlantic Ocean. The data demonstrate that the different cores along the transect have heterogenous geochemical and/or biomarker signatures and this is used to argue for different microbial assemblages across the transect, shaped by the presence of the salt diapir.
Main assessment
The data presented in this manuscript is of good quality and interesting. Especially the biomarker data as shown in figures 5&6 is extensive. The geochemical and/or biomarker data changes along the transect with core 78, from the methane seepage site, standing out. As such, the fundamental conclusion that the biogeochemistry/lipidomics, maybe related to the microbial assemblages, differ among the transect is supported by the data. It is likely that the salt diapir and associated methane seepages drives this. The presentation quality is good with clear language and figures.
However, in the current version of the manuscript the link between lipidomics and microbial assemblages is made to easy and with too much certainty. For example, the manuscript states that the lipid biomarkers indicate distinct ANME clades as shown by higher abundance of isoGDGT-1 to -3 in some cores. However, this is hard to see and normally indices such as the Methane Index would be used to explore the impact of ANME archaea in marine sediments (Zhang et al., 2011). The presence of isoGDGT-1 to -3 is not sufficient by itself as these lipids are also made by many other types of archaea. See recent review on GDGTs in marine sediments (Elling et al., 2026).
A much more nuanced discussion is needed on how biomarkers are used to infer microbial dynamics. This doesn’t mean that the differences between cores is not there, but the conclusions of what is driving this in terms of microbiology needs more nuance and expanded discussion as genomic data is lacking for these cores.
Secondly, it is not clear to me how the data presented in this submitted manuscript differs from that shown in a recent publication from this group (Redshaw et al., 2026). That published paper also appears to report biogeochemical and biomarker data from push cores across the same seep site. And the conclusion of this published paper “Spatial changes in the stratified system highlight the complex interplay of micro- and macro-seepage and provide insights into the seep’s evolution and impact on microbial dynamics across the carbonate structure” appears similar to the conclusion from this submitted manuscript. As such I can not determine whether the submitted manuscript represents a substantial contribution to scientific progress.
I encourage the authors to revise this manuscript and 1) provide a much more nuanced discussion about what the biomarker data represent in terms of microbial assemblages and 2) make it very clear what the novelty of this study is compared to published work.
David Naafs, 16th of September 2026.
Comments (in order of appearance)
Lines 103-118: An introduction into the (biomarker) methods is needed here, especially the wide range of biomarkers used in this manuscript. This includes iso and brGDGTs, chlorophyl, menaquinones, etc etc.
Lines 207-216: more details are needed here for the qTOF-MS methods. For example, what mobiles phases and gradients were used, what scan parameters (e.g. m/z range), etc etc.
Line 215-16: as only one internal standard was used for all different compounds, without response factors, the quantifications given here are semi-quantitative at best. Although this doesn’t undermine the results, this caveat needs to be clearly acknowledged.
Line 232: maybe I missed something, but I could not find methane concentrations in any of the figures. Methane data is key as this is proposed as main driver. So this data needs to be shown. Maybe in figure 2?
Lines 238-254 (and elsewhere): statistical proof needs to be given to support statements about parameters being different between cores. For example, is the TOC data in cores 15, 19, and 17 statistically different from that in cores 14 and 13 as stated? If it is, adding statistical data to support these statements will strengthen the manuscript. If not, then such passages need to be removed or altered. This applies to the whole manuscript.
Line 259: but for some the correlation (R2) is weak with values < 0.2. Is this still significant?
Line 262: See previous comment. Need to provide proof that these correlations are significant.
Figure 4: I wonder why no CH4 nor O2 data is presented here. Especially as CH4 is considered a key driver of some of the biomarker data. And are all cores anaerobic?
Line 307 (and throughout): crenarchaeol should not be called isoGDGT-5. Revise throughout the manuscript. IsoGDGT-5 has give cyclopentane rings and is normally not found in marine sediments.
Line 328: change “0-5 rings” to “0-4 cyclopentane rings as well as crenarchaeol”.
Line 338: no IPL versions of brGDGTs are found? As the authors argue that the brGDGTs are produced in the sediments and not transported from the terrestrial realm, wouldn’t IPL brGDGTs be expected?
Line 388-391: provide statistical evidence that the data from core 78 (the hole) is different from the other cores.
Lines 391-395: This sentence is not clear to me. If the methane has a d13C signature of – 71 ‰, I would expect the methanotrophic biomass to be even more depleted than that, but across all data, only 2 samples (2 and 8 cm from core 78) have d13Corg < -30 ‰. And even in those samples, d13Corg is around – 40 ‰. If methanotrophs are a major source of the carbon, wouldn’t d13Corg need to be even more depleted?
Line 404: if bioturbation is found, does that mean the core is not anaerobic?
Line 411: not clear why increased preservation can not be the main driver.
Line 436-437: why is degradation the reason and not reduced phytodetrital input? This requires more explanation
Line 446: thaumarchaeota have been renamed to Nitrososphaeria. Change throughout the manuscript.
Line 448: what core goes down to 800 cmbsf? All data is plotted down to 600 cmbsf, right?
Line 511-512: reference for these ANME thresholds are needed
Line 522-523: not clear why brGDGTs are assumed to be produced in the sediments and not transported from the terrestrial realm. Needs more discussion to rule out terrestrial production and transfer to marine realm. For example, use methods as proposed in (Sinninghe Damsté, 2016), maybe in combination with the BIT index (Hopmans et al., 2004). See also recent review on GDGTs in marine sediments (Elling et al., 2026).
Lines 529-532: not clear whether anybody has ever claimed brGDGTs to be derived from sulphate reducing bacteria, or is that not what this sentence means? So far the few known sources are aerobic (heterotrophic) acidobacteria (Sinninghe Damsté et al., 2018; Chen et al., 2022; Halamka et al., 2023).
References
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