| 04 Feb 2026
Holocene methane pockmarks in the Baltic Sea, Part I: Archaeal community composition based on tetraether lipids and 16S rRNA analysis
Abstract. Methane-rich pockmarks and shallow gas systems are prominent geomorphological features in the Baltic Sea that act as hotspots of microbial activity. Methane pockmarks in the Gdańsk Basin differ in seepage intensity, the efficiency of internal methane biofilters, and the influence of freshwater infiltration. The objective of this research was to examine the effects of methane seepage and submarine groundwater discharge (SGD) on the composition of archaeal communities and the archaeal tetraether lipids (GDGTs) produced by these communities across the examined gas systems. Additionally, the research assessed how these environmental factors affect the use and interpretation of GDGT-based proxies in such environments. The study investigates whether GDGT patterns in these gas systems primarily reflect methane-driven processes (anaerobic oxidation of methane and methanogenesis) or ammonia oxidation, which is a key process in the Baltic Sea. It also evaluates how reliably GDGT indices can be applied in this dynamic environment characterised by strong upward gas flow. The results show elevated GDGT concentrations in pockmark sediments compared with reference non-pockmark sediments; however, GDGT concentrations are variable and depend on whether the flow is active or inactive, reflecting episodic submarine groundwater discharge that coincides with methane release. Overall, GDGT concentrations are much higher at sites with minimal or no SGD. Nevertheless, consistently low Methane Index values (MI < 0.09), together with low GDGT-0/crenarchaeol (< 1) and GDGT-2/cren (< 0.04) ratios, indicate that the iGDGT patterns lack the typical enrichment associated with methane-rich and anaerobic oxidation of methane (AOM) settings, suggesting no strong AOM imprint on the GDGT pool. OH-GDGT% values are consistent with those of Baltic Sea surface sediments. GDGT-based proxies in this system, therefore, primarily reflect ammonia-oxidiser activity rather than methane flux. These findings highlight the complex interplay between SGD and methane fluxes in shaping archaeal communities, GDGT composition, and their sedimentary record. GDGT-based indices must be applied with caution in dynamic shallow gas systems.
This manuscript presents a multidisciplinary study integrating organic geochemistry (lipid biomarker analysis) with molecular biology (16S rRNA metabarcoding) to investigate microbial ecosystems associated with methane venting in the Baltic Sea under the influence of submarine groundwater discharge (SGD). The combined analysis of archaeal community composition and tetraether lipid distributions in methane pockmarks of the Gdańsk Basin is valuable and contributes to improving our understanding of how SGD-related processes—such as freshwater input, hydraulic pressure, and methane venting—may influence benthic archaeal communities.
However, the manuscript would benefit from clearer geochemical evidence supporting the influence of geofluids in the investigated cores. In particular, porewater data are not presented (I could not even find in the supplementary file), making it difficult for readers to assess whether there is a clear geofluid signal at different sediment depths or across spatially distinct sites. The fractional abundances of GDGTs and OH-GDGTs (Figs. 3 and 4) appear relatively similar among different sites and between the two cores at the same site (non-pockmark vs. pockmark). Without accompanying porewater profiles, it is challenging to evaluate the extent to which these sediments are directly influenced by geofluid input, especially given the typically heterogeneous nature of seepage systems.
Moreover, while bulk concentrations of GDGTs and OH-GDGTs differ among cores, these variations alone may not fully resolve whether the observed patterns reflect differences in microbial community input, total organic carbon enrichment, or varying terrestrial contributions. Additional geochemical context would greatly strengthen the interpretation of the lipid and microbial data.
With clearer presentation of porewater parameters and a more focused discussion linking GDGT indices to environmental conditions, the study has the potential to make a meaningful contribution to Biogeosciences. Hereby, I therefore recommend a major revision to further enhance the robustness and clarity of the manuscript.
Major comments.
Specific comments
Line 48: what is the implication of OH-GDGT% values consistent with those of Baltic Sea surface? What is the ratio and compared with what sampling sites? It is not clear for the OH-GDGT%
Line 50: what results are inferred for ammonia-oxidiser? Because high Crenarchaeol and thaumarchaeota genes? It should be stated clearly.
Line 353: it would be better to show the TOC results among different sediment cores. It is not sufficient to state the approach limit in Result section.
Line 379: FA is not necessary to be abbreviated from fractional abundance since it is not widely used, and it is easily confused with fatty acids (FA). In addition, fraction abundance can be marked with the symbol %.
Line 384: It is not necessary to show so many parameters in statistics. Only show r and p values and regardring Peasron or Spearman can be introduced in the Method section. Please check through the whole text.
Line 583: varrying should be varying.
Line 643: nutrients availability should be nutrient availability.
Line 648, Line 651, reference citations are wrong. Please check through accordingly.
Line 683: DSEG is an older phylogenetic name and now belong Thermoplasmatota. Please check the new phylogenetic lineages.
Line 723: Table 1 shows the GDGT-2/cren of 0.02 on average, but here it is maxim 0.4. It means some depth should be much lower than 0.02. please check the statement.
Fig. 1: It would be better to list the venting status in the figure for comparing the difference. For example, Ebullition, methangenesiis and refe for MET1, MET3, and MET4.
Fig. 2: why it a scatter plot for the TOC of MET3 rather than line curves like others? Please use a consistent plotting way.
Fig. 3: please adjust the scale of y axis in the down panel since the upper limit is not arrived to 0.06 and it doesn’t make to keep higher value of 0.08. It seems not necessary to plot minor compounds separately because it is still not clear.
Fig. 5. Same issue as Fig. 4. It is not necessary to separate OH-2 from other OH-GDGTs.
Fig. 6: It makes me confused about the presence of ANME-2a/2b. In the main text, it is stated that the higher ANME-2a/2b in the main text, Fig. 7, and supplementary Fig. S7 and S8. Why ANME-2a/2b abundance is not shown in Fig. 6.