Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Microbial communities provides both concrete degradation and self-healing. In this regard, studying of concrete microbial communities performed in the present study is urgent. The work contains all required data corresponding to the goal of the study. Therefore, the work may be accepted after minor revisions.

1. Two samples of concrete were heat-dried and stored. Could you emphasize the differences in microbial community composition between heat-dried-stored samples and fresh samples?
2. Table 3 can be supplemented. Please, add the column with the data on the mechanism of concrete healing / degradation for each microbial group.

Author Response

Comments 1: Two samples of concrete were heat-dried and stored. Could you emphasize the differences in microbial community composition between heat-dried-stored samples and fresh samples?

Response 1: We thank the reviewer for this important point. We have expanded both the results and discussion to more clearly distinguish heat-dried/stored samples (Samples 1-2) from freshly processed samples (Samples 3-7) and to describe how this may have influenced our observations. In the Results, we now note that Samples 1-2 showed lower alpha-diversity (and Sample 3 partially) relative to Samples 4-7 for both 16S and ITS profiles, while taxonomic profiles remained broadly overlapping across samples (Supplementary Tables 1-2), including multiple genera detected across all seven samples. In the Discussion, we explicitly address potential causes (including partial DNA degradation during drying/storage and preferential loss of low-abundance taxa) and emphasize that, despite reduced diversity, heat-dried samples did not form a distinct compositional outlier group and retained many dominant/recurrent concrete-associated taxa. Together, these additions clarify both the differences and the overall comparability between stored and fresh concrete samples. (Lines 193-198; 232-236; 303-315)

Comments 2: Table 3 can be supplemented. Please add a column with data on the mechanism of concrete healing/degradation for each microbial group.

Comments 2: We thank the reviewer for this constructive suggestion. We have revised Table 3 by adding a new column summarizing, for each microbial group, the principal mechanistic pathway(s) reported in the literature. For taxa where no concrete-specific activity has been reported, we explicitly indicate that the mechanism is unknown or that concrete-specific evidence remains limited. (Page 13)

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an emerging and interdisciplinary topic at the interface of microbiology and concrete engineering. While the study provides valuable descriptive insights into concrete-associated microbial communities, several revisions are recommended to strengthen its relevance, clarity, and applicability from a structural engineering perspective.The authors are encouraged to consider the following key revisions:
1. Clarify the Engineering Focus
   Clearly define the primary engineering objective of the study, and align the discussion and conclusions accordingly.
2- Expand the discussion on how the identified microbial communities may influence structurally relevant properties such as durability, crack development, serviceability, or long-term performance of concrete.
3. Explicitly state that microbial functional roles (e.g., degradation or biomineralization) are inferred from taxonomic identity and literature, and not experimentally verified within this study.
4. Discuss the limitations associated with the small number of samples and their representativeness of real structural concrete, particularly with respect to different concrete types and exposure conditions.
5. Provide a clearer assessment of how heat-drying and long-term storage may have influenced microbial diversity and comparability between samples.
6.Clarify key methodological aspects relevant to reproducibility, including PCR conditions, sequencing strategy, and data normalization prior to diversity analyses.
7. Report essential statistical indicators (e.g., NMDS stress values, envfit p-values and r²) to support the interpretation of multivariate analyses.
8. Specify the crack scales and serviceability conditions under which the discussed microbial self-healing mechanisms may be relevant from an engineering standpoint.

Author Response

Comments 1: Clarify the Engineering Focus. Clearly define the primary engineering objective of the study, and align the discussion and conclusions accordingly.

Response 1: We thank the reviewer for this helpful suggestion. We have clarified the primary engineering objective of the study in the introduction and aligned the discussion and conclusions accordingly. Specifically, we now state explicitly that our goal is to identify resident taxa capable of colonizing urban concrete, thereby providing a shortlist of organisms that may serve as minimally engineered starting points for future biotechnological applications. (Lines 74-80; 298-303; 429-431)

Comments 2: Expand the discussion on how the identified microbial communities may influence structurally relevant properties such as durability, crack development, serviceability, or long-term performance of concrete.

Response 2: We thank the reviewer for this valuable suggestion. We have expanded the discussion to more explicitly connect the identified concrete-associated communities to structurally relevant performance concepts, including durability, crack development/serviceability, and long-term performance. Specifically, we added text clarifying that microbe–concrete interactions may influence performance primarily by altering near-surface and crack-network permeability/ion transport and local microenvironments: acid-driven decalcification and persistent biofilms can increase porosity and potentially accelerate ingress of aggressive agents, whereas biomineralization-driven CaCO₃ precipitation may partially seal microcracks and reduce permeability under wetting conditions, thereby slowing downstream deterioration mechanisms. (Lines 364-376)

Comments 3: Explicitly state that microbial functional roles (e.g., degradation or biomineralization) are inferred from taxonomic identity and literature, and not experimentally verified within this study.

Response 3: We thank the reviewer for this important point. We have now made this explicit in the revised manuscript by clarifying that the functional roles discussed are inferred from amplicon-based taxonomic assignments and published literature, and were not experimentally validated in this study. (Lines 388; 421-423)

Comments 4: Discuss the limitations associated with the small number of samples and their representativeness of real structural concrete, particularly with respect to different concrete types and exposure conditions.

Response 4: We thank the reviewer for this important comment. We have expanded the discussion to explicitly acknowledge limitations related to the small sample size and the representativeness of the sampled materials relative to structural concrete. We now clarify that our samples capture a limited subset of urban concrete types and exposure histories, and that variation in mix design could substantially influence microbiome composition and function. Accordingly, we frame our findings as an initial taxonomic baseline and shortlist of resident taxa, and we outline future work using larger, stratified sampling across concrete classes and exposure conditions to test generality and quantify drivers. (Lines 404-421)

Comments 5: Provide a clearer assessment of how heat-drying and long-term storage may have influenced microbial diversity and comparability between samples.

Response 5: We thank the reviewer for this important point. We have revised the results and discussion to provide a clearer assessment of how heat-drying and long-term storage may have influenced microbial diversity and comparability relative to freshly processed samples. In the results, we now explicitly report that alpha-diversity was lower in the heat-dried/stored samples (and partially Sample 3) for both 16S and ITS profiles, consistent with the possibility that drying/storage reduces detectable richness and evenness, particularly for low-abundance taxa. In the discussion, we expand on plausible mechanisms, including partial DNA degradation and preferential loss of rare templates, as well as potential compositional bias from differential survival of DNA from more resilient taxa. Importantly, we also highlight that overall community composition remained broadly comparable across sample types: heat-dried samples did not form distinct compositional outliers, and Supplemental Tables 1-2 show substantial overlap of classified taxa across all samples, including multiple genera detected in all seven samples alongside sample-specific enrichments present in both heat-dried and fresh concrete. Together, these additions support a balanced interpretation: heat-drying/storage may reduce apparent diversity and bias detection of rare taxa, but the dominant and recurrent concrete-associated taxa remain robustly detectable, enabling qualitative comparability for the primary aim of identifying resident candidate genera. (Lines 193-198; 232-236; 303-315)

Comments 6: Clarify key methodological aspects relevant to reproducibility, including PCR conditions, sequencing strategy, and data normalization prior to diversity analyses.

Response 6: We thank the reviewer for this important suggestion. We have revised the methods to improve reproducibility by clarifying key experimental and analytical parameters. We report the detailed PCR reaction setup, clarify the sequencing strategy, and explicitly state the data-handling procedures prior to diversity analyses. (Lines 121-125)

Comments 7: Report essential statistical indicators (e.g., NMDS stress values, envfit p-values and r²) to support the interpretation of multivariate analyses.

Response 7: We thank the reviewer for this suggestion. The statistical indicators for envfit (r² and p-values) are provided in Supplementary Table 4. We have revised the results text to explicitly report the NMDS stress value and to direct readers to Supplementary Table 4 and Supplementary Figure 3 for the full multivariate statistics. (Line 290)

Comments 8: Specify the crack scales and serviceability conditions under which the discussed microbial self-healing mechanisms may be relevant from an engineering standpoint.

Response 8: We thank the reviewer for this suggestion. We have revised the discussion to more explicitly state the crack scales and serviceability conditions under which microbially mediated self-healing mechanisms are most relevant. (Lines 404-421)