Assembly and Source of the Lithobiontic Microbial Community in Limestone

Round 1

Reviewer 1 Report

General comments

Valid research that fits the scope of either the journal, or the special issue. Several detail is missing, and all the specific comments need to be addressed before publication

 

Specific comments

Line 16. “Due to its unique rock properties”. Which properties, dissolution along fractures and abundance of CO₃ in solution that favour life of microbial communities? Please, be more specific

Line 41. Specify type of community

Line 53. Mention (to rise the impact of your research) the importance of karst rocks worldwide. You need to say that karst aquifers of carbonate sedimentary origin, underlie a land surface covering approximately 15% of the globe and supply about 25% of the world’s population with potable water. Refer to the papers below that discuss the concept, and provide the worldwide maps of karst:

- Goldscheider, N.; Chen, Z.; Auler, A.S.; Bakalowicz, M.; Broda, S.; Drew, D.; Hartmann, J.; Jiang, G.; Moosdorf, N.; Stevanovic, Z.; et al. Global distribution of carbonate rocks and karst water resources. Hydrogeology Journal 2020, 28, 1661–1677

- Medici, G., Lorenzi, V., Sbarbati, C., Manetta, M., Petitta, M. 2023. Structural Classification, Discharge Statistics, and Recession Analysis from the Springs of the Gran Sasso (Italy) Carbonate Aquifer; Comparison with Selected Analogues Worldwide. Sustainability, 15(13), p.10125.

 

Line 115. Specify the two to three specific objectives of your research by using numbers (e.g., i, ii, iii)

Line 168. More detail on the t-test that are very common in natural sciences. Which is the purpose? Why did you use them? Briefly explain the mathematical background

Line 170. “kruskal()”, typo here?

Line 287. “Acid corrosion”. I would not use this term for a natural process, please reward it

Line 309. Which characteristics? Geochemical composition? Roughness in correspondence of fractures? Please, be more specific

Line 382. I would add another sentence as concluding remark

Line 403. Please add the relevant literature suggested above

 

Figures and tables

Figure 5. Increase fonts on axes, letters and figures difficult to read

Moderate editing for the language are required 

Author Response

Response to Reviewer #1

• General comments

 [Comment 1]: Valid research that fits the scope of either the journal, or the special issue. Several detail is missing, and all the specific comments need to be addressed before publication

[Response]: Thanks for the reviewer’s positive and insightful comments. These comments, together with those listed below enabled us to have a deeper thinking on this issue, and thus guided us to conduct a thorough revision of the original manuscript. Detailed modifications please see our responses to the following comments.

The entire manuscript has been polished by native English speakers. Please refer to the attachment for proof of the polishing.

• Specific comments

[Comment 2]: Line 16. “Due to its unique rock properties”. Which properties, dissolution along fractures and abundance of CO3 in solution that favour life of microbial communities? Please, be more specific

[Response]: A very insightful comment indeed. In fact, what we are trying to express here is that the porous nature, relatively rough texture, and high calcium and magnesium content of limestone make it susceptible to microbial colonization. Therefore, we have included relevant information in our manuscript to address these characteristics of limestone (Page 1, line 16).

 

[Comment 3]: Line 41. Specify type of community

[Response]: Wonderful comment! We are indeed describing the microbial community, and we acknowledge the limited research on the process of microbial community assembly (Page 2, line 44).

 

[Comment 4]: Line 53. Mention (to rise the impact of your research) the importance of karst rocks worldwide. You need to say that karst aquifers of carbonate sedimentary origin, underlie a land surface covering approximately 15% of the globe and supply about 25% of the world’s population with potable water. Refer to the papers below that discuss the concept, and provide the worldwide maps of karst:

- Goldscheider, N.; Chen, Z.; Auler, A.S.; Bakalowicz, M.; Broda, S.; Drew, D.; Hartmann, J.; Jiang, G.; Moosdorf, N.; Stevanovic, Z.; et al. Global distribution of carbonate rocks and karst water resources. Hydrogeology Journal 2020, 28, 1661–1677

- Medici, G., Lorenzi, V., Sbarbati, C., Manetta, M., Petitta, M. 2023. Structural Classification, Discharge Statistics, and Recession Analysis from the Springs of the Gran Sasso (Italy) Carbonate Aquifer; Comparison with Selected Analogues Worldwide. Sustainability, 15(13), p.10125.

[Response]: Very constructive comment! We have incorporated the reviewer's suggestion and have included additional information in our manuscript (Page 2, line 58-61) regarding the global ecological significance of karst landscapes, particularly with regard to water resource availability. However, due to restrictions on accessing global-scale data on karst distribution, we have decided not to present maps containing relevant information in the manuscript.

 

[Comment 5]: Line 115. Specify the two to three specific objectives of your research by using numbers (e.g., i, ii, iii)

[Response]: Thank you for the reviewer's comment! We have made the necessary changes in the manuscript (Page 4, line 117, 118, 119 and 121) by replacing the Arabic numerals in the corresponding sections with Roman numerals.

 

[Comment 6]: Line 168. More detail on the t-test that are very common in natural sciences. Which is the purpose? Why did you use them? Briefly explain the mathematical background

[Response]: The t-test is a statistical method used to compare the degree of difference in means between two groups of data. It employs the t-distribution theory to infer the probability of observing such differences and determine whether the difference between the two means is statistically significant. Permutation t-test relaxes the assumptions of normality and homoscedasticity, making it suitable for comparing significance between fungi and bacteria originating from the same source (Page 4, line 185-187).

 

[Comment 7]: Line 170. “kruskal()”, typo here?

[Response]: Good comment! The format you mentioned is not a printing error. It is a common expression in the R programming language, where functions are typically written as “function name” + “()”.

 

[Comment 8]: Line 287. “Acid corrosion”. I would not use this term for a natural process, please reward it

[Response]: Very good comment! In the natural environment, it is not appropriate to use acid corrosion as the primary mechanism, as acid rain occurrences are relatively rare. Our intention was to express that the metabolic byproducts generated after biotic colonization of rocks disrupt the rock structure. Therefore, we will rephrase the aforementioned content in the manuscript (Page 13, line 304-306).

 

[Comment 9]: Line 309. Which characteristics? Geochemical composition? Roughness in correspondence of fractures? Please, be more specific

[Response] The term " characteristics " refers to porosity, permeability, roughness, and chemical composition. We have provided an explanation of these properties in the manuscript (Page 13, line 327).

 

[Comment 10]: Line 382. I would add another sentence as concluding remark

[Response]: It is an excellent suggestion, and we have provided a prospect for future research at the end of the conclusion (Page 15-16, line 402-407).

 

[Comment 11] Line 403. Please add the relevant literature suggested above

[Response]: We have appropriately cited the references mentioned in the comments at their respective positions (Page17, line 447-452).

• Figures and tables

[Comment 12]: Figure 5. Increase fonts on axes, letters and figures difficult to read

[Response]: Excellent feedback! We have increased the font size of the axes in Figure 5 for better readability.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors presented the interesting paper on the influence of various factors on the process of colonization of limestone sand by microorganisms from the environment.

In the Introduction, it is necessary to add information that heterotrophic microorganisms (bacteria and microscopic fungi) dissolve minerals during the so-called heterotrophic leaching (or chemoorganotrophic leaching), which can include two main mechanisms: acidolysis and complexolysis (Sand and Bock, 1991). These mechanisms can also be accompanied by redoxolysis, a change in the redox potential of the environment by bacteria, which affects the extraction and mobility of metals that make up minerals.

Lines 74-76 - they can survive, but we are talking about settling. Those, a new habitat on the surface of the mineral must offer some advantages.

Line 119 - decipher the abbreviations of various processing (despite the fact that they are deciphered in the Annotation).

The Methods should describe how limestone came into contact with microbial sources (decaying wood, soil and a third source (requiring explanation)). This is one of the key issues and should be described in detail.

Line 259 - again we are talking about unknown sources. The authors have an assumption - what are these sources?

Author Response

[Comment 1] The authors presented the interesting paper on the influence of various factors on the process of colonization of limestone sand by microorganisms from the environment.

[Response]: Thanks for the reviewer’s positive and insightful comments. These comments, together with those listed below enabled us to have a deeper thinking on this issue, and thus guided us to conduct a thorough revision of the original manuscript. Detailed modifications please see our responses to the following comments.

 

[Comment 2] In the Introduction, it is necessary to add information that heterotrophic microorganisms (bacteria and microscopic fungi) dissolve minerals during the so-called heterotrophic leaching (or chemoorganotrophic leaching), which can include two main mechanisms: acidolysis and complexolysis (Sand and Bock, 1991). These mechanisms can also be accompanied by redoxolysis, a change in the redox potential of the environment by bacteria, which affects the extraction and mobility of metals that make up minerals.

[Response]: The insightful comment provided has enhanced the coherence of the introduction section. We have fully incorporated the reviewer's statement and appropriately cited the references in the manuscript at relevant positions (Page 2,line 48-51).

 

[Comment 3] Lines 74-76 - they can survive, but we are talking about settling. Those, a new habitat on the surface of the mineral must offer some advantages.

[Response]: Very constructive feedback! Indeed, our choice of words was inappropriate. Therefore, we will modify "survive" to "settling" to better reflect the actual state (Page 3, line 84).

 

[Comment 4] Line 119 - decipher the abbreviations of various processing (despite the fact that they are deciphered in the Annotation).

[Response]: Good comment! In fact, we have already provided an explanation of the abbreviations in the abstract section (Page 1, line 21-22). However, for the sake of readability, we have also included this explanation in the Introduction section (Page 4, line 114-116).

 

[Comment 5] The Methods should describe how limestone came into contact with microbial sources (decaying wood, soil and a third source (requiring explanation)). This is one of the key issues and should be described in detail.

[Response]: Good comment! In the experimental site (Figure S1), apart from air and rainfall, our samples were placed in an open environment where the potential microbial sources mainly included the surrounding environment. Firstly, decaying wood, rich in cellulose and humic sub-stances, provided an ideal habitat for microbial colonization. The placement of decaying wood at elevated positions facilitated the dispersion of microorganisms residing on the wood through wind transport. Secondly, a concrete fence encircled the area at a distance of approximately 1 meter above the ground, hosting abundant lichens with fungal spores that could potentially disperse via wind. Lastly, scattered soil particles in the vicinity of the site could also become a source of microbial population through wind-mediated dispersal to the experimental samples (Page 5, line 133-142).

 

[Comment 6] Line 259 - again we are talking about unknown sources. The authors have an assumption - what are these sources?

[Response]: A highly insightful comment indeed! One of the issues addressed in this study is the origin of rock-dwelling microorganisms. To investigate this, we incorporated decaying wood, the concrete fence, and surrounding soil as potential sources. The results indicated that approximately 30-40% of the microorganisms originated from these three sources. As for the remaining unidentified sources, we speculate that they may originate from the air and rainfall. The microbial content in the air and rainfall will be a focus of our future investigations.