Integrated Assessment of Benthic Bacterial Community Physiology, Structure, and Function Across C, N, P, and S Gradients in Lake Villarrica Sediments, Chile

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript ‘An integrated physiological, structural and functional assessment of benthic bacterial community across C, N, P, and S gradients in Lake Villarrica Sediments, Chile’ is devoted to the study of bacterial communities in lake sediments with different nutrient exposition.

Multiple replicates and the use of different analysis methods and statistics result in a large dataset that is the strength of this article. The weakness of the article is English, which requires editing. Some sentences should be reformulated, as the meaning is not clear. I would recommend rewriting the Results (section 3). This section is replete with data that the authors have already provided in the tables. Sentences with too many numbers are cumbersome and difficult to read and comprehend. The manuscript can be published after English editing.

1. There is a problem with the numbering of sections in methods.
2. Please provide the name of the institution in English: “Then, approximately 800 g of sediment replicates were aseptically placed into sterile plastic bags, kept at 4 °C, and transported to the Laboratorio de Investigación Interdisciplinaria en Microbiología Aplicada (LIMA) at the Universidad Católica de Temuco (UCT)”
3. Tables 1 and 2: The meaning of the superscripts for ABCD is unclear.
4. Section 4.3. Information about the number of ASVs is more logical to move forward before information about phyla.
5. The coordinates of the lake are repeated twice (in Introduction and 2.1. lake description).
6. Section 2.4. Why are some substance names capitalized? ”However, the classification into functional categories of N sources from the PM3B (Ammonia, Nitrite, Nitrate, Amino acids and Derivatives, Amines and Derivatives, Amides and Derivatives, Nucleobases and Derivatives, and Peptides), P sources from the PM4A (Phosphate, Polyphosphate, Hypo and Thiophospate, Phophomonoesters, Phosphodiesters, Phosphotriesters, Phosphonates, Inositol Phosphate, and Other Po Compounds) and S sources from the PM4A (Sulfates, Thiosulfates, Tetrathionates, Thio and Dithiophosphates, S-amino acids and derivatives, and Other So Compounds) microwell plates, was established according to their molecular structure and inorganic or organic origin.”

In some sentences, substances are indicated with a capital letter, followed by a small one in the sentence. Section 4.2. “This was especially true for organic P compounds, including Phosphonates, Other Po Compounds, and Phosphomonosters, over inorganic Pi. It has been observed that waste water sludges preferentially utilize phosphomonoesters…” In addition, the phosphomonoesters in these two sentences are written differently.

7. The “general ASVs” mentioned in section 3.4 are illustrated in Figure 5, which is difficult for interpretation. I recommend to use a Venn diagram instead, as it is a standard and convenient for this type of data, even for five groups.
8. The authors' descriptions lack consistency. For example, in the sentence “Similarity in ASVs was highest in the groupings VB × NL (419 ASVs) and VB × SL × NL” a number is provided for the first group but omitted for the second.
9. Section 4.2. Cyanobacteria is Cyanobacteriota now (https://gtdb.ecogenomic.org/).
10. Section 4.2. “The preference for these C groups over glucides may be due to the greater availability of simple sugars in the higher nutrient sites, with a lower probability of being utilized, but also due to differences in bacterial community structures and function [24,67].” It is not clear what it refers to “with a lower probability of being utilized.” Glucides?
11. Section I Introduction “Under these conditions, the REDOX series proceeds through exhaustion of electron donors, shifting microbial community structure and function in a complex series of coupled processes (further described below) that act to shape the stoichiometry of nutrients in the overlying water.” – the meaning is unclear, reformulate, please.
12. Figure 8 – “The bacterial families most related to the variables are listed. “Do these factors not affect the other families? It seems there are no anthropogenic effects on the microbial communities. Also, why are F4 and F6 indicated in the figure if they show no relation to the variables?
13. I think it is not good to start the title of the article with an indefinite article.

Comments on the Quality of English Language

1. “Similarity in ASVs was highest in the groupings”. Superlative form of high is the highest.
2. “Additionally, SRB from hypoxic sediment samples of Dangdong Bay, South Korea, were observed to change their functioning in response to oxygen availability [79], suggesting REDOX coupling may be important for S utilization in Lake Villarrica, where Si metabolization was higher in sites with lower DO in (e.g., VL).” The last preposition “in” is not required.
3. “Interestingly, the less impacted site, NL, reported higher abundances…” Sediments cannot report. Please, rephrase.
4. section 4.2. “the metabolism of different bacterial communities” change to “the metabolism of these bacterial communities” (as we are discussing your communities); “in the low nutrient sites” change to “at the low nutrient sites” etc; "higher biomasses were observed” biomass – uncountable noun in this context, “biomass was observed”

The text needs to be corrected because of the number of comments like this.

Author Response

Reviewer 1.

Comments and Suggestions for Authors

The manuscript 'An integrated physiological, structural and functional assessment of benthic bacterial community across C, N, P, and S gradients in Lake Villarrica Sediments, Chile' is devoted to the study of bacterial communities in lake sediments with different nutrient exposition.

1. Multiple replicates and the use of different analysis methods and statistics result in a large dataset that is the strength of this article. The weakness of the article is English, which requires editing. Some sentences should be reformulated, as the meaning is not clear. I would recommend rewriting the Results (section 3). This section is replete with data that the authors have already provided in the tables. Sentences with too many numbers are cumbersome and difficult to read and comprehend. The manuscript can be published after English editing.
2.  

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We consider presenting numerical results in the text necessary for contextualizing the reader, but we also understand that filling this section with too many numbers and comparisons can be challenging. For this reason, we have exhaustively revised the whole manuscript, shortening long paragraphs (especially in the Results section). At the same time, in this study, two native English speakers (Dr. Jaisi and Dr. Spears) were directly involved in the writing of the manuscript. Finally, to improve the manuscript's writing, we used the Grammarly program.

 

3. There is a problem with the numbering of sections in methods.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. In effect, Section 2, "Materials and methods," had problems with the numbering. We suitably corrected the problem.

 

4. Please provide the name of the institution in English: "Then, approximately 800 g of sediment replicates were aseptically placed into sterile plastic bags, kept at 4 °C, and transported to the Laboratorio de Investigación Interdisciplinaria en Microbiología Aplicada (LIMA) at the Universidad Católica de Temuco (UCT)"

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. Although the name is a proper name that should be written in Spanish, we agree with the reviewer that using it in English could have a greater impact. We resolved this as "Interdisciplinary Research Laboratory in Applied Microbiology" in lines 164-165.

 

5. Tables 1 and 2: The meaning of the superscripts for ABCD is unclear.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The superscript letters (ABCD) indicate significant differences obtained from a one-way ANOVA with Tukey's honest significant difference (HSD) test among sample sites for each measurement. It was clarified and added in lines 309-311 and lines 380-382.

 

6. Section 4.3. Information about the number of ASVs is more logical to move forward before information about phyla.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. However, in section 4.3, "Quantification of the total bacterial community and functional nutrient-cycling gene," we discuss the abundance of nutrient-cycling genes, not ASVs.

 

7. The coordinates of the lake are repeated twice (in Introduction and 2.1. lake description).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We remove this information from section 2.1 to avoid redundancy.

 

8. Section 2.4. Why are some substance names capitalized?" However, the classification into functional categories of N sources from the PM3B (Ammonia, Nitrite, Nitrate, Amino acids and Derivatives, Amines and Derivatives, Amides and Derivatives, Nucleobases and Derivatives, and Peptides), P sources from the PM4A (Phosphate, Polyphosphate, Hypo and Thiophospate, Phophomonoesters, Phosphodiesters, Phosphotriesters, Phosphonates, Inositol Phosphate, and Other Po Compounds) and S sources from the PM4A (Sulfates, Thiosulfates, Tetrathionates, Thio and Dithiophosphates, S-amino acids and derivatives, and Other So Compounds) microwell plates, was established according to their molecular structure and inorganic or organic origin."

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have corrected this inconsistency and homogenized all substrate categories to lowercase to avoid confusion throughout the manuscript.

 

9. In some sentences, substances are indicated with a capital letter, followed by a small one in the sentence. Section 4.2. "This was especially true for organic P compounds, including Phosphonates, Other Po Compounds, and Phosphomonosters, over inorganic Pi. It has been observed that waste water sludges preferentially utilize phosphomonoesters…" In addition, the phosphomonoesters in these two sentences are written differently.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. In line with the above observation, we have standardized the substrate names and appropriately labeled phosphomonoesters in both cases. Lines 571-572.

 

10. The "general ASVs" mentioned in section 3.4 are illustrated in Figure 5, which is difficult for interpretation. I recommend to use a Venn diagram instead, as it is a standard and convenient for this type of data, even for five groups.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. However, we consider that an UpSet plot provides a more accurate and interpretable representation of shared and unique ASVs among multiple groups. We tested the Venn diagrams and found them visually complex and challenging to interpret when more than three sets are involved. In our UpSet plot, we used a matrix-based layout that scales efficiently with the number of groups. This approach allowed us to visualize intersection sizes more clearly, facilitated quantitative comparisons, and avoided overlapping-area distortions that occurred with the Venn diagram. Therefore, we believe that the UpSet plot offers a more robust and standardized alternative for summarizing ASV intersections among five sampling sites.

 

11. The authors' descriptions lack consistency. For example, in the sentence "Similarity in ASVs was highest in the groupings VB × NL (419 ASVs) and VB × SL × NL" a number is provided for the first group but omitted for the second.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We corrected the sentence by adding the missing data in lines 425-426.

 

12. Section 4.2. Cyanobacteria is Cyanobacteriota now (https://gtdb.ecogenomic.org/).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The Cyanobacterial phylum name was suitably changed to Cyanobacteriota throughout the manuscript.

 

13. Section 4.2. "The preference for these C groups over glucides may be due to the greater availability of simple sugars in the higher nutrient sites, with a lower probability of being utilized, but also due to differences in bacterial community structures and function [24,67]." It is not clear what it refers to "with a lower probability of being utilized." Glucides?

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The sentence was rewritten for a more understandable paragraph in lines 549-552.

 

14. Section I Introduction "Under these conditions, the REDOX series proceeds through exhaustion of electron donors, shifting microbial community structure and function in a complex series of coupled processes (further described below) that act to shape the stoichiometry of nutrients in the overlying water." – the meaning is unclear, reformulate, please.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The sentence was rewritten for a more understandable paragraph in lines 63-65.

 

15. Figure 8 – "The bacterial families most related to the variables are listed. "Do these factors not affect the other families? It seems there are no anthropogenic effects on the microbial communities. Also, why are F4 and F6 indicated in the figure if they show no relation to the variables?

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The factors shown in Figure 8 highlight the strongest correlations with certain bacterial families, but this doesn't mean that other families were unaffected; their responses were weaker or less direct. We kept F4 and F6 in the figure to show the full spatial distribution of all sampling sites, even though their relationship with the main variables was not significant.

 

16. I think it is not good to start the title of the article with an indefinite article.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The title was adjusted accordingly to the reviewer's suggestion.

 

17. Comments on the Quality of English Language

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. In the present study, two native English speakers (Dr. Jaisi and Dr. Spears) were directly involved in the writing of the manuscript. Additionally, we used the Grammarly program to improve the writing further.

 

18. "Similarity in ASVs was highest in the groupings". Superlative form of high is the highest.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We corrected the sentence by adding the missing data from lines 425-426.

 

19. "Additionally, SRB from hypoxic sediment samples of Dangdong Bay, South Korea, were observed to change their functioning in response to oxygen availability [79], suggesting REDOX coupling may be important for S utilization in Lake Villarrica, where Si metabolization was higher in sites with lower DO in (e.g., VL)." The last preposition "in" is not required.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We corrected the whole sentence in lines 588-591.

 

20. "Interestingly, the less impacted site, NL, reported higher abundances…" Sediments cannot report. Please, rephrase.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We rephrased the sentence from lines 399-400.

 

21. Section 4.2. "the metabolism of different bacterial communities" change to "the metabolism of these bacterial communities" (as we are discussing your communities); "in the low nutrient sites" change to "at the low nutrient sites" etc; "higher biomasses were observed" biomass – uncountable noun in this context, "biomass was observed"

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We rephrased the sentences from lines 567-574.

 

22. The text needs to be corrected because of the number of comments like this.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have revised the manuscript carefully based on your comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study surveys benthic bacterial communities in Lake Villarrica (Chile) across five sites presumed to span a nutrient impact gradient. You measure sediment chemistry (TC/TOC/OM, TN, TP, TS), run CLPPs with EcoPlates and PM plates, quantify total bacteria and 10 functional genes by qPCR, and profile community composition via 16S metabarcoding with FAPROTAX and PICRUSt2 predictions. You conclude that the most impacted site (VB) has higher nutrients and total bacteria but lower physiological activity and lower abundances for several nutrient-cycling genes; low-impact sites look more versatile. Unfortunately, at this stage the manuscript is not still acceptable for publication, and a major revision is needed.

 

General comments

Line numbering (important): the PDF has no continuous line numbers, which made line-by-line feedback harder. Please resubmit a line-numbered manuscrip. In my notes below I use section cues and figure/table references—please map these to proper line numbers in revision.

Overall verdict: The topic is relevant and the multi-layer dataset is interesting. However, there are major issues that affect scientific soundness and readability:

Sediment chemistry units/magnitudes look wrong in Table 1 (e.g., very low TC/TOC values coexisting with double-digit % OM at VB). This undermines the central gradient and any downstream stats based on these numbers. Full audit of units, recalculation, and figure/table re-generation are required.

Replication and statistics. It’s unclear whether the “quadruplicate replicate samples” are truly independent cores per site. If subsamples from a single dredge, many ANOVAs are pseudo-replicated. Please clarify the sampling unit and re-analyze accordingly (mixed models or site-level aggregation).

Methods clarity. Several details are missing/ambiguous for CLPP (media recipes, dye usage across plates, incubation temperature rationale) and qPCR (standards, efficiencies per assay, LOD/LOQ). These need tightening for reproducibility.

Functional annotations. KEGG IDs are mis-assigned in places (e.g., pqqC shown with K10944—the amoA KO). Correct all KO mappings and update the figure/text accordingly.

Alpha-diversity/labels. Supplementary ST4 labels “Observed ASVs” with values that are actually reads, not ASVs. Fix labels and any derived stats.

Language and consistency. Numerous broken words/typos (e.g., “Desvet” for SD; “OPR” vs ORP; PoT vs PoP; spacing artifacts) need a full editorial pass.

 

Title & Abstract

Title and abstract: remove broken words/hyphenation artifacts; report key effect sizes with units and exact P-values for main claims (e.g., VB vs others on nutrients, AWCD, and gene abundances).

 

Introduction & Study rationale

Clarify the a priori site order (NL < PuB < PoP < SL < VB) and then test it against corrected chemistry. Add simple plots (e.g., z-scores) to show the gradient after the unit audit.

 

Where you quote lake loading/impact numbers, add converted units and references with uncertainty, not only Tg yr⁻¹ style statements. (Same section).

 

Materials & Methods

Sampling/replication

Specify clearly: number of independent cores per site, core diameter, top-layer thickness (e.g., 0–5 cm?), spacing between cores, and how subsamples were generated. State whether in situ DO/ORP were measured in overlying water or sediments; current DO reported as “%” for VB (~1.5 %) needs unit clarity (saturation % vs mg L⁻¹).

 

Sediment chemistry

Major: Audit units/magnitudes in Table 1. Values cited in the text (VB “almost twice” others) and comparisons to literature don’t reconcile with plausible TC/TOC given OM ~14 % at VB. Re-run any stats/plots that depend on these data.

Fix the EPA method reference: text cites 3015A for sediments, while the reference list correctly lists 3051A for sediments/sludges/oils. Align text and references with the right method and matrix.

 

CLPP

Provide full CM9/NM9/PM9/SM9 recipes (salts, pH, ionic strength), and justify adding TTC to PM3B/PM4A while EcoPlates already contain a redox dye. Note how you ensured OD comparability across plate types. Include a plate map for PM3B/PM4A and your functional category mapping (machine-readable in Supplementary).

Incubation at 30 °C: give an ecological rationale versus in-situ temperatures; otherwise consider showing a sensitivity check or temper interpretation.

Report AWCD both as endpoint and AUC (more robust to timing), and normalize category means by number of substrates per category.

 

DNA/qPCR

Confirm freeze–thaw is −80 °C/95–96 °C (96 °C is unusual); cite evidence that this pre-treatment does not bias composition. State template for standards, amplicon sizes, efficiency and R² per assay, and LOD/LOQ. Report abundances per g dry weight (state moisture basis). Replace “Desvet” with SD.

 

Metabarcoding & functional prediction

Rarefaction depth is 92,178 reads; in ST4, the “Observed ASVs” column lists six-figure numbers that are actually reads. Correct labels; ensure alpha indices are computed on ASVs. Provide NSTI distribution for PICRUSt2 and discuss limits in sediments.

 

KO mappings: fix pqqC (should be K06131, not K10944 which is amoA). Update Fig. 7, legend, and text; then revisit conclusions that depend on these predictions.

 

Statistics

If subsamples are nested within site, use site as the unit or apply mixed-effects (random = core/plate). Report assumption checks and effect sizes. For β-diversity, specify transformation (e.g., Hellinger/CLR) before RDA; report axis eigenvalues, adjusted R², permutation ANOVA. Avoid statements like “nutrients account for 88.42 % of variance” without model stats.

 

Results

Chemistry

After the unit audit, provide medians and ranges (sediment chemistry is often skewed). Clarify whether ORP was measured in sediments or water; current values look strongly negative.

 

CLPP

Keep Fig. 2 endpoints, but add AUC per plate/site with CIs (Supplementary). In Fig. 3, add the number of substrates per category and show category-normalized AWCD.

 

qPCR

Add an assay performance table (efficiency, R²) and check the surprising patterns (e.g., relatively high mcrA at both NL and VB). If archaeal contribution is expected, discuss that 16S quantification here is bacterial.

 

Community composition

In Fig. 4–5 legends, give taxonomic rank for clades like “Sva0485”, “KD4-96”, “Subgroup_17”, and confirm UpSet counts are ASVs.

 

Functional prediction

After fixing KO IDs, quantify qPCR vs PICRUSt2 concordance per gene across sites (e.g., Spearman ρ). Temper claims when predictions disagree with qPCR.

 

RDA

Report permutation ANOVA for axes, variance partitioning if you claim nutrient vs gene effects, and transformation used. Re-phrase the “88.42 %” statement to standard ordination language.

 

Discussion

 

Revisit all comparisons to the literature after chemistry corrections (e.g., TP ranges, TC/TOC vs OM). Right now the narrative on how “VB is almost twice others” conflicts with plausible units.

CLPP interpretation: separate “preference/activity on plates” from ecological function; discuss potential dye inhibition/temperature mismatch. Cite any controls performed (none described).

N-cycle inference (low amoA, variable nosZ): consider adding NO₃⁻/NH₄⁺/N₂O data or sediment incubations in future work; keep current causal claims cautious.

P-cycle: be careful proposing pqqC as a PSB marker given KO mis-annotation and non-significant site differences by qPCR. Suggest enzyme assays (e.g., phosphatase) or metatranscriptomics as next steps.

 

Major revisions (must-fix)

Chemistry unit/magnitude audit (TC/TOC/OM, TN, TP, TS), with full QA/QC (CRMs, duplicates, recoveries), and re-analysis of all dependent results/figures.

Clarify replication and re-analyze statistics to avoid pseudo-replication (state unit, adopt mixed models or site-level tests).

Strengthen CLPP methods and reporting (media recipes, dye rationale, 30 °C justification, category normalization, plate maps in Supplementary).

Fix alpha-diversity labeling in ST4 (reads vs ASVs) and ensure metrics are computed on ASV tables.

Quantify qPCR vs PICRUSt2/FAPROTAX agreement and temper functional claims where discordant.

Language/formatting pass to resolve typos, broken words, units, and abbreviation inconsistencies.

 

Minor revisions (examples)

“electro conductivity” → “electrical conductivity.”

Clarify TOC method (acidification/combustion) with the exact standard; ensure OM is derived consistently.

State TTC concentration and background correction for dye self-reduction.

Table 2 caption: show n, SD, and per-assay efficiency/R² in Supplementary.

Fig. 4 legend: include rank for clades (Sva0485, KD4-96, Subgroup_17, etc.).

 

Author Response

Reviewer 2.

Comments and Suggestions for Authors

The study surveys benthic bacterial communities in Lake Villarrica (Chile) across five sites presumed to span a nutrient impact gradient. You measure sediment chemistry (TC/TOC/OM, TN, TP, TS), run CLPPs with EcoPlates and PM plates, quantify total bacteria and 10 functional genes by qPCR, and profile community composition via 16S metabarcoding with FAPROTAX and PICRUSt2 predictions. You conclude that the most impacted site (VB) has higher nutrients and total bacteria but lower physiological activity and lower abundances for several nutrient-cycling genes; low-impact sites look more versatile. Unfortunately, at this stage the manuscript is not still acceptable for publication, and a major revision is needed.

Response: We thank the reviewer for their exhaustive revisions of our manuscript. We have addressed all comments in the best possible way. We hope to meet the reviewer's expectations and publish our work in this prestigious journal.

 

General comments

1. Line numbering (important): the PDF has no continuous line numbers, which made line-by-line feedback harder. Please resubmit a line-numbered manuscrip. In my notes below I use section cues and figure/table references—please map these to proper line numbers in revision.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The line numbering was added.

 

Overall verdict: The topic is relevant and the multi-layer dataset is interesting. However, there are major issues that affect scientific soundness and readability:

2. Sediment chemistry units/magnitudes look wrong in Table 1 (e.g., very low TC/TOC values coexisting with double-digit % OM at VB). This undermines the central gradient and any downstream stats based on these numbers. Full audit of units, recalculation, and figure/table re-generation are required.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. After a complete review of units, we identified that the correct units for TC and TOC are % rather than mg g⁻¹. Additionally, we noticed that TN was initially reported in mg g⁻¹ and not % dry weight. We converted the values ​​(× 0.1) and checked the consistency between OM, TC, TOC, and TN: the ratios OM ≈ 1.724×TOC and molar C/N ≈ 9–12 are now consistent with typical values ​​for lake sediments. After correcting TN (× 0.1), all TC, TOC, and TN values are plausible for a nutrient-saturated system. No additional scaling is needed for dependent figures such as RDA, since the factor is kept at the same magnitude, so the figure remains unchanged. The table is replaced accordingly to the reviewer's observations.

 

3. Replication and statistics. It's unclear whether the "quadruplicate replicate samples" are truly independent cores per site. If subsamples from a single dredge, many ANOVAs are pseudoreplicated. Please clarify the sampling unit and re-analyze accordingly (mixed models or site-level aggregation).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The quadruplicate replicate samples are independent, not from a single dredge. We performed the sampling as described in Campos et al. (2021; doi:10.1016/j.scitotenv.2021.145782). In this article, we explain in detail the sampling following the US Environmental Protection Agency (EPA) recommendations. We decided to resume the information by citing the article that fully describes the procedure. We added the concept "(homogeneous and individual)" to clarify it in lines 159-160. Also, because replicates are individual rather than pseudoreplicates, the statistical analysis does not need to be corrected.

 

4. Methods clarity. Several details are missing/ambiguous for CLPP (media recipes, dye usage across plates, incubation temperature rationale) and qPCR (standards, efficiencies per assay, LOD/LOQ). These need tightening for reproducibility.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The required information was added to the Supplementary Material (Supplementary Table ST5. Primer sets and PCR conditions used for the quantification of bacterial functional genes).

 

5. Functional annotations. KEGG IDs are mis-assigned in places (e.g., pqqC shown with K10944—the amoA KO). Correct all KO mappings and update the figure/text accordingly.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The KEGG ID for pqqC and soxB were incorrectly written in the text, but not in the figure. Now is correct, as the reviewer observed, in lines 475 and 480.

 

6. Alpha-diversity/labels. Supplementary ST4 labels "Observed ASVs" with values that are actually reads, not ASVs. Fix labels and any derived stats.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. In effect, the reviewer is correct. Supplementary Table 4 (now Supplementary Table ST6, Observed ASVs) was incorrectly provided as reads rather than ASVs. This was a mistake that we have already resolved. Now, with the new data, we run again the statistics and no significant difference (P < 0.05) was observed among sample sites.

 

7. Language and consistency. Numerous broken words/typos (e.g., "Desvet" for SD; "OPR" vs ORP; PoT vs PoP; spacing artifacts) need a full editorial pass.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. All the broken words and typos were corrected in the manuscript, figures, and tables.

 

Title & Abstract

8. Title and abstract: remove broken words/hyphenation artifacts; report key effect sizes with units and exact P-values for main claims (e.g., VB vs others on nutrients, AWCD, and gene abundances).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The broken words/hyphenation artifacts are generated automatically by the journal template; however, we removed them to improve the presentation. Numerical comparison was added to the abstract, but only for nutrient, CLPP, and gene abundance, as the reviewer recommended, while also keeping the limit of words as mandated by the journal for this section.

 

Introduction & Study rationale.

9. Clarify the a priori site order (NL < PuB < PoP < SL < VB) and then test it against corrected chemistry. Add simple plots (e.g., z-scores) to show the gradient after the unit audit.

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We incorporate the analysis recommended by the reviewer after unit correction and sentences relating to material and methods (lines 148-149), results (lines 301-305), and discussion lines 509-510).

 

10. Where you quote lake loading/impact numbers, add converted units and references with uncertainty, not only Tg yr⁻¹ style statements. (Same section).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. However, the official information about nutrient inputs to the Lake Villarrica is just the cited ones (https://planesynormas.mma.gob.cl/archivos/2020/proyectos/Informe_Ufro_Evaluacion_de_Medidas_de_N_y_P_como_Anexo_a_Minuta_Carga_Critica.pdf). In a previous article (https://doi.org/10.1007/s00248-023-02173-2), we utilized the same report with units in Mg year⁻¹). In the current manuscript, we wanted only to simplify the information by presenting it as Tg yr⁻¹.

 

Materials & Methods

Sampling/replication

11. Specify clearly: number of independent cores per site, core diameter, top-layer thickness (e.g., 0–5 cm?), spacing between cores, and how subsamples were generated. State whether in situ DO/ORP were measured in overlying water or sediments; current DO reported as "%" for VB (~1.5 %) needs unit clarity (saturation % vs mg L⁻¹).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. As we explained before, we followed the methodology described in Campos et al. 2021 (doi:10.1016/j.scitotenv.2021.145782) and the US Environmental Protection Agency (EPA) recommendations. However, we added the thickness of superficial sediment in line 159. The in situ measurement was performed on the sediment sample, not on the water, and the DO for VB is ok, effectively 1.5%. We corrected the DO unit to % throughout the manuscript.

 

Sediment chemistry

12. Major: Audit units/magnitudes in Table 1. Values cited in the text (VB "almost twice" others) and comparisons to literature don't reconcile with plausible TC/TOC given OM ~14 % at VB. Re-run any stats/plots that depend on these data.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have responded to this in the previous observation. We also presented these results and comparisons in a more measured way.

 

13. Fix the EPA method reference: text cites 3015A for sediments, while the reference list correctly lists 3051A for sediments/sludges/oils. Align text and references with the right method and matrix.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The method in the text was suitably corrected, in lines 181-182.

 

CLPP

14. Provide full CM9/NM9/PM9/SM9 recipes (salts, pH, ionic strength), and justify adding TTC to PM3B/PM4A while EcoPlates already contain a redox dye. Note how you ensured OD comparability across plate types. Include a plate map for PM3B/PM4A and your functional category mapping (machine-readable in Supplementary).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We now provide complete CM9/NM9/PM9/SM9 formulations in the supplementary material (Supplementary Table ST1) and clarify that EcoPlates™ already include a tetrazolium redox dye, whereas PM3B/PM4A do not; therefore, we added TTC (0.01% w/v) to PM3B/PM4A to enable respiration readouts in the manuscript. In the PM3 and PM4 plates protocol, to measure the cell-mediated metabolism of the chemicals, one of two proprietary color-generating systems (Cat. #74351 Biolog Redox Dye Mix MA) needs to be added to all wells (lines 191-194).

 

The Optical Density (OD) value for each well was corrected by subtracting the control (blank well) value from the corresponding plate well, as described by Sofo and Ricciuti (2019; doi:10.3390/app9194035) (line 204). OD comparability across plate types was ensured by a fixed wavelength (590 nm), identical read geometry and incubation conditions, blank subtraction per plate, and category means normalized by the number of substrates, as recommended in the cited article.

 

We also include a complete Ecoplate, PM3B, and PM4A plate map, as well as machine-readable functional category mapping, in the Supplementary Material (Supplementary Figure SF2) and a CVS archive (DOI: 10.5281/zenodo.17436230).

 

15. Incubation at 30 °C: give an ecological rationale versus in-situ temperatures; otherwise consider showing a sensitivity check or temper interpretation.
16.  

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We're really sorry about this. Our incubations were at 25 °C, not 30 °C, which is closer to the in situ temperature. By incubating lake sediment bacterial communities at 25 °C, we established a physiologically relevant temperature for mesophilic bacteria, a key functional group in sediment biogeochemistry that thrives in the 20–45 °C range. The experimental temperature, therefore, simulates a plausible seasonal maximum to which the bacterial community is adapted. Furthermore, using this temperature accelerates the metabolic activity and community turnover, allowing us to effectively study microbial dynamics and biogeochemical processes within a practical timeframe.

 

Please see Bååth and Kritzberg (2024) (doi: 10.1007/s00248-024-02353-8) for studies on lake communities where increased temperatures are better at stimulating bacterial growth than lower temperatures. In any case, as we did not perform this experiment at the in situ temperature, we have tempered our interpretations, stating the limitations of this approach in the discussion section (lines 526-534).

 

17. Report AWCD both as endpoint and AUC (more robust to timing), and normalize category means by number of substrates per category.

 

Response: We sincerely appreciate the reviewer's suggestion to include both endpoint and AUC-based AWCD analyses. We recognize that the AUC metric can provide a more integrated representation of metabolic activity over time. However, in the present study, we focused on the endpoint AWCD (after 96 h of incubation), as this approach is widely standardized and comparable across CLPP studies. Maintaining a single, well-defined endpoint allows consistent interpretation with previous reports on aquatic sediments using Biolog PM plates. Additionally, the temporal kinetics of color development were homogeneous across sites and were already normalized by the number of substrates, minimizing potential bias from timing effects. We have clarified this rationale in the revised Methods section and cited the relevant literature supporting endpoint-based AWCD as a practical and accepted approach.

 

The AUC values (Supplementary Table ST3) confirmed site-specific differences in metabolic dynamics. Although VB showed relatively high AUC due to early color development, its final AWCD remained the lowest among sites, indicating an early but limited overall metabolic response. We included this justification in lines 324-327.

 

 

DNA/qPCR

18. Confirm freeze–thaw is −80 °C/95–96 °C (96 °C is unusual); cite evidence that this pre-treatment does not bias composition. State template for standards, amplicon sizes, efficiency and R² per assay, and LOD/LOQ. Report abundances per g dry weight (state moisture basis). Replace "Desvet" with SD.

 

Response: We appreciate the reviewer's observation. The freeze–thaw procedure was performed using a water bath, alternating between −80 °C (freezing) and 65 °C (thawing). The 96 °C value originally reported was incorrect; in practice, the water bath was maintained at 65 °C, a standard temperature for promoting cell lysis without compromising DNA integrity. The Methods section has been corrected accordingly (line 225). The word "Desvet" has already been changed to SD, as we explained in an earlier comment. We have added standard template reporting for each assay, including primer sequences, amplicon sizes, standard type and range, calibration parameters (slope, efficiency, R²), and LOD/LOQ definitions and values. All qPCR assays met the accepted performance criteria (slope = −3.1 to −3.6, efficiency = 90–110 %, R² ≥ 0.98). LODs ranged between 1–6 copies·reaction⁻¹ and LOQs within the linear range (2–367 copies·reaction⁻¹) in the Supplementary Material, as we explained in an above response. Slightly over-efficient assays (nosZ and mcrA) remained within acceptable limits and showed consistent linearity and single-peak melting curves.

 

Metabarcoding & functional prediction

19. Rarefaction depth is 92,178 reads; in ST4, the "Observed ASVs" column lists six-figure numbers that are actually reads. Correct labels; ensure alpha indices are computed on ASVs. Provide NSTI distribution for PICRUSt2 and discuss limits in sediments.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. In effect, the reviewer is correct. We resolved this mistake accordingly, based on the reviewer's observation, as we explained in an above response.

 

Additionally, we have included a density plot of NSTI scores in the Supplementary Material (Supplementary Figure 4), as requested. The mean NSTI score is 0.3246, reflecting the microbial community's phylogenetic distance. Sediment microbiomes typically contain many uncultured, poorly represented lineages, so their NSTI values are naturally higher than those of host-associated or soil communities. Please let us know if further changes are needed. We introduced NSTI information in the results and discussion to temper interpretations of PICRUST results (lines 452-455) and discussion (lines 714-721).

 

20. KO mappings: fix pqqC (should be K06131, not K10944 which is amoA). Update Fig. 7, legend, and text; then revisit conclusions that depend on these predictions.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The KEGG ID for pqqC and soxB were incorrectly written in the text, but not in the figure. The conclusion doesn't depend on this mistake. The mistake has now been corrected in lines 475 and 480.

 

Statistics

21. If subsamples are nested within site, use site as the unit or apply mixed-effects (random = core/plate). Report assumption checks and effect sizes. For β-diversity, specify transformation (e.g., Hellinger/CLR) before RDA; report axis eigenvalues, adjusted R², permutation ANOVA. Avoid statements like "nutrients account for 88.42 % of variance" without model stats.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. As we explained before, each replicate is an independent and homogeneous sediment sample, not a pseudoreplicate. For this reason, statistical analysis does not need correction. The PCoA, as already explained in lines 264-265. It was obtained and tested for significance by the Kruskal−Wallis test and PERMANOVA based on Bray−Curtis distances. For the RDA, the explanation is already given in lines 278-280 using Hellinger distances. We created an additional table for supplementary material (Supplementary Table ST7) containing the requested data for the RDA. We tempered the interpretation of the RDA because OM, TN, TP, and TC were not statistically significant (p > 0.05), likely due to collinearity with total sulfur (TS), which was significant. These covariates were retained in the ordination plot to illustrate their negative association with functional genes. The corresponding paragraph has been revised accordingly (lines 482–484).

Results

Chemistry

22. After the unit audit, provide medians and ranges (sediment chemistry is often skewed). Clarify whether ORP was measured in sediments or water; current values look strongly negative.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We assessed the unit audit as the reviewer recommended deriving changes for TC, TOC, and TN. Also, as we explained before, the in situ analysis was performed to the sediment sample, which is why OPR is reported as strongly negative.

 

CLPP

23. Keep Fig. 2 endpoints, but add AUC per plate/site with CIs (Supplementary). In Fig. 3, add the number of substrates per category and show category-normalized AWCD.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have kept Figure 2 showing the endpoint AWCD values (96 h) for clarity and comparability with prior CLPP studies, and we have now calculated and reported the corresponding AUC values with 95% confidence intervals for each site in Supplementary Table S2. This information was added in lines 204-207, 315-316, and 324-328.

 

In addition, we modified Figure 3 to include the amount of substrates per functional category. The category-normalized mean AWCD values are already considered in the heatmap. We hope that these changes enhance comparability among categories and improve the robustness of interpretation.

 

qPCR

24. Add an assay performance table (efficiency, R²) and check the surprising patterns (e.g., relatively high mcrA at both NL and VB). If archaeal contribution is expected, discuss that 16S quantification here is bacterial.

 

25. Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The primer set described in Cisex et al. (2023; doi:10.3390/microorganisms11030660) used in our study is specific to bacteria; hence, the contribution of archaea is not expected. We discussed that, indeed, mcrA abundances in our sediment are lower than in other eutrophic lakes; the elevated abundance in NL and VB may be due to increasing TOC and OM content. This is exposed in lines 604-610.

 

Community composition

26. In Fig. 4–5 legends, give taxonomic rank for clades like "Sva0485", "KD4-96", "Subgroup_17", and confirm UpSet counts are ASVs.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have added additional information to clarify the reviewer's notes. The paragraphs now state as "uncultured delta proteobacterium Sva0485 in line 407, 419", "), Subgroup_17 (family, Acidobacteriota) in lines 405 and 417", and " unclassified group Chloroflexia KD4-96 in lines 406 and 419. Regarding the UpSet analysis, they effectively are ASVs, not OTUs.

 

Functional prediction

27. After fixing KO IDs, quantify qPCR vs PICRUSt2 concordance per gene across sites (e.g., Spearman ρ). Temper claims when predictions disagree with qPCR.

 

Response: We appreciate the reviewer's suggestion regarding the quantitative comparison between qPCR and PICRUSt2 predictions. In the revised version, we have ensured that our interpretation of PICRUSt2 results is presented cautiously and in a moderate tone, highlighting consistencies with qPCR trends when evident and explicitly avoiding overinterpretation where discrepancies may occur. We already stated the limitations of PICRUSt. In any case, we have been more explicit in cases when this is evident, for example, the pqqC gene in the paragraph written in lines 714-721.

 

RDA

28. Report permutation ANOVA for axes, variance partitioning if you claim nutrient vs gene effects, and transformation used. Rephrase the "88.42 %" statement to standard ordination language.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have already addressed these observations above.

 

Discussion

29. Revisit all comparisons to the literature after chemistry corrections (e.g., TP ranges, TC/TOC vs OM). Right now the narrative on how "VB is almost twice others" conflicts with plausible units.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The discussion was revised to align with the existing studies. These changes were introduced in lines 506-519.

 

30. CLPP interpretation: separate "preference/activity on plates" from ecological function; discuss potential dye inhibition/temperature mismatch. Cite any controls performed (none described).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We discussed and prepared a paragraph clarifying that preference/activity on plates is just an approach under laboratory conditions to the real ecological functions, and that the possible inhibitory effect of TTC and differences in temperature in lines 526-534 are to be considered.

 

31. N-cycle inference (low amoA, variable nosZ): consider adding NO₃⁻/NH₄⁺/N₂O data or sediment incubations in future work; keep current causal claims cautious.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript and the recommendation for future work. Indeed, we are currently working on sediment mesocosm experiments that consider some of these variables. We expect to release the study in the near future.

 

32. P-cycle: be careful proposing pqqC as a PSB marker given KO mis-annotation and non-significant site differences by qPCR. Suggest enzyme assays (e.g., phosphatase) or metatranscriptomics as next steps.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript and the recommendation for future work. We have smoothed the sentence about pqqC accordingly, avoiding proposing it as a molecular marker for PSB. The KO mis-annotation was corrected, as explained before. We are glad about the reviewer's proposal for enzyme assays and metatranscriptomics, as we have already implemented some approaches in Lake Villarrica and other representative lakes. We expect to share these results in the near future.

 

Major revisions (must-fix)

33. Chemistry unit/magnitude audit (TC/TOC/OM, TN, TP, TS), with full QA/QC (CRMs, duplicates, recoveries), and re-analysis of all dependent results/figures.

 

Response: We have already addressed these issues in previous responses.

 

34. Clarify replication and re-analyze statistics to avoid pseudo-replication (state unit, adopt mixed models or site-level tests).

 

Response: We have already addressed these issues in previous responses.

 

35. Strengthen CLPP methods and reporting (media recipes, dye rationale, 30 °C justification, category normalization, plate maps in Supplementary).

 

Response: We have already addressed these issues in previous responses.

 

36. Fix alpha-diversity labeling in ST4 (reads vs ASVs) and ensure metrics are computed on ASV tables.

 

Response: We have already addressed these issues in previous responses.

 

37. Quantify qPCR vs PICRUSt2/FAPROTAX agreement and temper functional claims where discordant.

 

Response: We have already addressed these issues in previous responses.

 

38. Language/formatting pass to resolve typos, broken words, units, and abbreviation inconsistencies.

 

Response: We have already addressed these issues in previous responses.

 

Minor revisions (examples)

39. "electro conductivity" → "electrical conductivity."

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have corrected this in line 307.

 

40. Clarify TOC method (acidification/combustion) with the exact standard; ensure OM is derived consistently.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have added a sentence to clarify it in lines 176-179.

 

41. State TTC concentration and background correction for dye self-reduction.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We added extra information about TTC concentrations in lines 191-194 and discussed possible interferences in the discussion section, as recommended by the reviewer.

 

42. Table 2 caption: show n, SD, and per-assay efficiency/R² in Supplementary.

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. The "n" is explained in the footnotes, alright. Desvet was corrected to SD as the reviewer previously recommended, and efficiency/R² are in the supplementary material (Supplementary Table ST5).

 

43. 4 legend: include rank for clades (Sva0485, KD4-96, Subgroup_17, etc.).

 

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We added this paragraph to the legend "Taxonomic ranks for placeholder clades are indicated: Sva0485 (family, Anaerolineaceae), KD4-96 (order, Chloroflexota), Subgroup_17 (family, Acidobacteriota). Classification follows the SILVA 138.1 database" (lines 418-421).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for your revision, the paper is much improved. Most major points are addressed (units, methods, statistics, and clearer interpretation). A few small fixes remain before acceptance:
Residual internal inconsistencies.

Fig. 2 caption still says “incubated at 30 °C” while Methods specify 25 °C; please fix it.

Gene label consistency: “ppqC” appears in the Fig. 7 caption/table headers; standardize to pqqC everywhere (and search the whole document).

Site code: Table 1 row shows “PoT” instead of “PoP”; fix to match all other sections.

Sampling detail still a bit thin. You assert replicates are independent, but the Methods still lack core diameter, lateral spacing between cores, and exact surface layer thickness used for analyses (<10 cm is given, but please specify the interval, e.g., 0–5 cm). Please fix it.

Chemistry distribution reporting. Means ± SD are fine, but given sediment skew, reporting medians and ranges (even in Supplementary) would strengthen the robustness of the comparisons.

qPCR vs PICRUSt2 concordance. You softened interpretation (good), but you did not add the simple concordance analysis originally suggested (e.g., Spearman ρ across sites per gene between qPCR and PICRUSt2). A small Supplementary table/plot would close this loop.

Minor template artifacts persist. The PDF still shows a few broken words and “h ps” style URL spacing in the front matter; a final proof pass will clear these.

Author Response

Response to the Reviewers/Editor Comments Second Round

 

Reviewer 2.

Thanks for your revision, the paper is much improved. Most major points are addressed (units, methods, statistics, and clearer interpretation). A few small fixes remain before acceptance:

Residual internal inconsistencies.

 

1. 2 caption still says “incubated at 30 °C” while Methods specify 25 °C; please fix it.

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have corrected the mistake in Line 319.

 

2. Gene label consistency: “ppqC” appears in the Fig. 7 caption/table headers; standardize to pqqC everywhere (and search the whole document).

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have corrected the mistakes in lines 384, 504, 463, and 726

 

3. Site code: Table 1 row shows “PoT” instead of “PoP”; fix to match all other sections.

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have resolved the mistake in the table.

 

4. Sampling detail still a bit thin. You assert replicates are independent, but the Methods still lack core diameter, lateral spacing between cores, and exact surface layer thickness used for analyses (<10 cm is given, but please specify the interval, e.g., 0–5 cm). Please fix it.

Response: We appreciate the reviewer’s observation. We want to be sorry if the information is not clear. On this occasion, we have provided more information to make it more understandable. The Methods section has been revised to specify that sediments were collected using a Petersen-type grab, as described by Campos et al. [34]. Each replicate corresponded to the upper sediment layer (< 10 cm), and samples were collected approximately 5 m apart to ensure spatial independence. We can state the exact thickness for replicates, since we use a Petersen-type grab and not core sampling. A new paragraph was generated in lines 158-162.

 

5. Chemistry distribution reporting. Means ± SD are fine, but given sediment skew, reporting medians and ranges (even in Supplementary) would strengthen the robustness of the comparisons.

Response: We thank the reviewer for this valuable suggestion. Given the potential non-normal distribution of sediment chemistry variables, we have now included the corresponding medians and ranges (minimum–maximum) for all parameters (TC, TOC, OM, TN, TP, TS) in the Supplementary Table ST1. Means ± SD remain reported in Table 1 for consistency and comparability with previous studies. We also add the following paragraph to the footnote of Table 1: “Corresponding medians and ranges (min–max) for each variable are provided in Supplementary Material ST1 to account for potential non-normality in sediment chemistry distributions.” in lines 315-317.

 

6. qPCR vs PICRUSt2 concordance. You softened interpretation (good), but you did not add the simple concordance analysis originally suggested (e.g., Spearman ρ across sites per gene between qPCR and PICRUSt2). A small Supplementary table/plot would close this loop.

Response: We thank the reviewer for this constructive suggestion. We added a Spearman’s rank correlation analysis between qPCR-measured and PICRUSt2-predicted gene abundances across sampling sites. The results (Supplementary Table ST8) show moderate to strong concordance (ρ = 0.63–0.88, p < 0.05) for most genes, supporting that PICRUSt2 predictions broadly reflect the qPCR-based trends, while we maintained a conservative interpretation given the inherent limitations of functional inference from 16S data. We added information about this analysis in the materials and methods (lines 293-295), results (lines 488-490), and discussion (lines 702-705).

 

7. Minor template artifacts persist. The PDF still shows a few broken words and “h ps” style URL spacing in the front matter; a final proof pass will clear these.

Response: We sincerely appreciate the present reviewer's contribution to improving the quality of our manuscript. We have revised the whole manuscript, looking for spacing and broken words. We hope to have resolved all of them.