Are There Benefits from Thermal Bacteria for Health? The Hydrogenome Role
, Daniel Glez-Peña 4,5, Carla Miranda 3,6, Patrícia Poeta 3,6, João Coutinho 7, Florentino Fdez-Riverola 4,5, Ana Torrado-Agrasar 8,9, María Luisa Rúa 8,9 and Gilberto Igrejas 1,2,3,*Round 1
Reviewer 1 Report
The authors investigate the presence and positive effect of bacterial species in 5 analyzed natural thermal / mineral waters. The chemical composition and proportion of individual identified bacterial species were analysed. All in all, my opinion is that the paper is interesting, it belongs to the content of the scope and aim of this Journal, it is well written, but some parts need to be better explained (in a clearer way) and after changes it could be accepted for publication in the journal.
Realistically this is a moderate revision, but since there is no such option I choose a major revision.
My comments are as follows:
The title of the paper mentions the term only thermal water, and throughout the manuscript two types of water are mentioned on the one hand thermal and on the other mineral. Namely, thermal water can also be mineral, and mineral does not have to be thermal water. You also explained in lines 136-137 that in Portuguese hot springs water is referred to as natural mineral water. However, you are now writing a paper in English and using English terminology and need to adapt to the language. Look for the definition of thermal water by temperature, and is the sample from Portugal marked with PT_SA thermal or mineral water? You stated in line 142-143 that it contains a lot of CO2 so this is also a confirmation of mineral water.
Please uniform this throughout the all text.
In the introduction, everything is focused on thermal waters, nothing on mineral waters. Maybe to explain the difference and similarities ....
Also the paper is written in two parts, the first dealing with the physicochemical composition and the second dealing with bacterial communities. It would be very interesting if you could link the physico-chemical composition of water with the occurrence of a population of bacterial communities.
In lines 122-123 you stated the number and richness of thermal waters in Spain and that some of them are used for therapeutic purposes. In the paper, clearly state why you chose three waters from Spain and two from Portugal. Which was the guiding thread? Why these waters?
Title of pater: termal vs. thermal
Line 32: “In each location, 50L of water were collected to physicochemical characterization and….” - It may be more clever to simplify: In collected water sample….
Line 33: Try not to use abbreviations in Abstract (NGS9, explain it as in line 97.
Line 37: write the sulfur compounds in parentheses correctly, as 2-, not -2
Line 44: Rrna correct to rRNA
Lines 109-115: This part should be either simplified or excluded. It stands out, that is, it does not fit best with the rest of the Introduction.
Lines 122: Divide a large sentence into two, the second would begin as follows: In this Spanish province ....
In section 2.2. it would be good to list the exact locations or show the locations on a map to make it clearer to readers.
Sampling time and years should also be stated, I mean month and year.
In Table 1. Sulphur S(2-) change to sulphide (S2-)
Line 164: sulfates, sulfates change to sulfates, sulphides
For example, in the Abstract you state the compound "RS" (line 39), in the results and discussion you state the compound "RS and C" (line 313), also in Conclusion (line 492), however in the Table there is no result of the analysis of these compounds.
Line 303: Write the chemical formulas of anionic compounds in the correct way.
For PC1-PC4, list the main components considered.
The resolution of Figure 5 is very poor, unacceptable, not visible to read.
Author Response
Reviewers #1:
Comments and Suggestions for Authors:
“The authors investigate the presence and positive effect of bacterial species in 5 analyzed natural thermal / mineral waters. The chemical composition and proportion of individual identified bacterial species were analysed. All in all, my opinion is that the paper is interesting, it belongs to the content of the scope and aim of this Journal, it is well written, but some parts need to be better explained (in a clearer way) and after changes it could be accepted for publication in the journal.
Realistically this is a moderate revision, but since there is no such option I choose a major revision.”
The authors acknowledge the comment made by the reviewer.
The manuscript has been revised taking into consideration the reviewer’s comments.
“My comments are as follows:
The title of the paper mentions the term only thermal water, and throughout the manuscript two types of water are mentioned on the one hand thermal and on the other mineral. Namely, thermal water can also be mineral, and mineral does not have to be thermal water. You also explained in lines 136-137 that in Portuguese hot springs water is referred to as natural mineral water. However, you are now writing a paper in English and using English terminology and need to adapt to the language. Look for the definition of thermal water by temperature, and is the sample from Portugal marked with PT_SA thermal or mineral water? You stated in line 142-143 that it contains a lot of CO2 so this is also a confirmation of mineral water.
Please uniform this throughout the all text.”
Considering the definition of natural mineral waters by the European legislation and the Medical Hydrology, added in the Introduction section, and that all waters used in this study have health properties, we adopted for all samples the terminology of thermal waters or more correctly, natural thermal mineral waters. All manuscript was revised in this line [line 29, 63-73, 158-162, 244-245, 246, 265, 301, 305, 317, 346, 521, Table 1 and 2, Figure 1].
“In the introduction, everything is focused on thermal waters, nothing on mineral waters. Maybe to explain the difference and similarities ....”
This information was added [line 63-73].
“Also the paper is written in two parts, the first dealing with the physicochemical composition and the second dealing with bacterial communities. It would be very interesting if you could link the physico-chemical composition of water with the occurrence of a population of bacterial communities.”
There are few studies that analyze the physiochemical characteristics together with the microbial communities existents of these water or the association between microorganism composition and the therapeutic indications. For instance, Paduano et al. (2018) reported that the anti-rheumatic properties of sulphurous-bromine-iodine thermal water have associated with the presence of the followed bacteria belonging to genera Geothermobacterium, Thermus, Syntrophomonas Desulfomonile, Thiofaba and Thermodesulfovibrio, as described above in the manuscript [line 108-111].
“In lines 122-123 you stated the number and richness of thermal waters in Spain and that some of them are used for therapeutic purposes. In the paper, clearly state why you chose three waters from Spain and two from Portugal. Which was the guiding thread? Why these waters?”
As mentioned in the manuscript, both chosen regions are made up of numerous hot springs. In addition, this work is the result of a partnership between a Portuguese University and a Spanish University. Considering these factors, the geographic proximity of these hot springs to the institutions and their characteristics, five of these waters were selected.
“Title of pater: termal vs. thermal”
The title was revised [line 3-4].
“Line 32: “In each location, 50L of water were collected to physicochemical characterization and….” - It may be more clever to simplify: In collected water sample….”
The sentence was rewritten [line 34-36].
“Line 33: Try not to use abbreviations in Abstract (NGS9, explain it as in line 97.”
The sentence was rewritten [line 34-36].
“Line 37: write the sulfur compounds in parentheses correctly, as 2-, not -2”
The compounds were changed [line 41].
“Line 44: Rrna correct to rRNA”
The word was changed [line 48].
“Lines 109-115: This part should be either simplified or excluded. It stands out, that is, it does not fit best with the rest of the Introduction.”
This paragraph was revised [line 131-136].
“Lines 122: Divide a large sentence into two, the second would begin as follows: In this Spanish province ....”
This sentence was divided [line 145].
“In section 2.2. it would be good to list the exact locations or show the locations on a map to make it clearer to readers.”
This information was added [line 172-175]. (o ideal seria remeter esta informação para o outro artigo que está na Frontiers)
“Sampling time and years should also be stated, I mean month and year.”
This information was added [line 172-175]. (o ideal seria remeter esta informação para o outro artigo que está na Frontiers)
“In Table 1. Sulphur S(2-) change to sulphide (S2-)”
The word was changed [Table 1].
“Line 164: sulfates, sulfates change to sulfates, sulphides”
The sentence was revised [line 190].
“For example, in the Abstract you state the compound "RS" (line 39), in the results and discussion you state the compound "RS and C" (line 313), also in Conclusion (line 492), however in the Table there is no result of the analysis of these compounds.”
These abbreviations were added in Table 1 and revised in the manuscript (C corresponds to conductivity and DR corresponds to dry residue) [line 42, 340, 526, Table 1].
“Line 303: Write the chemical formulas of anionic compounds in the correct way.”
The compounds were changed [line 330].
“For PC1-PC4, list the main components considered.”
Principal component analysis is a dimensionality-reduction method that is often used to reduce the dimensionality of large data sets, by transforming a large set of variables into a smaller one that still contains most of the information in the large set. In this study, the PC analysis included all physicochemical parameters described in Table 1 and Figure 3, as mentioned in the manuscript in section 2.3 of Material and Methods. This information was also reformed in the Results section [line 287-288]. (penso que será isto a que o revisor se refere)
“The resolution of Figure 5 is very poor, unacceptable, not visible to read.”
The Figure 5 was divided [Figure 5 and 6] and the followed table was renumbered.
Reviewer 2 Report
The manuscript discusses a unique type of water, thermal waters, which are often used for thermal bathing. The manuscript was verbose in some areas, but not overly problematic. The methods section would benefit from explaining the methods used for assessing the chemical parameters. There were sections of the manuscript that were difficult to read and an additional English review would be beneficial. Figure 5 is a particularly important figure for the review; however, the font in the figure was too small for reading or interpreting. The manuscript pertains to sources of water that do have public exposure, and for that reason, greater concern or appreciation for the public health implications of this work are recommended. Beyond the NGS results from the microorganisms, is there any health relevance to the physiochemical paramaters? Several parameters exceed drinking water guidelines, although this is not for drinking water – maybe some of these levels should be of concern for public health?
Title: The title is not grammatically correct for standard English. Alternative titles: “Does the hydgrogenome suggest that thermal contain bacteria beneficial for health?” “Are there benefits from thermal bacteria for health? Lessons from the hydrogenome”
Line 29: characterize (spelling)
Line 32: replace “to” with “for” , insert “to” before analyse.
Line 36-38: reword lines
Line 39: communities is possessive rather than plural, so community’s
Line 42: “allowing the safe use” is leading reader to believe that all these waters are presumably safe. An alternative writing would provide greater margin for risk from harmful microbes and harmful chemicals that may be present that were not captured by these brief snapshots. Rather than “allowing the safe use”, it may be “increasing the likelihood of safe use”
Line 57: “knew” is leading statement. Better verb choice maybe “surmised”
Line 66: What reference supports that therapeutic purpose of each water is linked to microbial diversity rather than minerals? Need to maybe mention more clearly in this statement that the mineral composition is influenced by the microbial diversity.
Line 100: Compresses or ‘comprises’
*Stopped at line 100 with English editing.
The overall NGS results and related information appears appropriate content for Water. The scientific method used was primarily exploratory in nature with no specific hypotheses.
Author Response
Reviewers #2:
Comments and Suggestions for Authors:
“The manuscript discusses a unique type of water, thermal waters, which are often used for thermal bathing. The manuscript was verbose in some areas, but not overly problematic. The methods section would benefit from explaining the methods used for assessing the chemical parameters. There were sections of the manuscript that were difficult to read and an additional English review would be beneficial. Figure 5 is a particularly important figure for the review; however, the font in the figure was too small for reading or interpreting. The manuscript pertains to sources of water that do have public exposure, and for that reason, greater concern or appreciation for the public health implications of this work are recommended. Beyond the NGS results from the microorganisms, is there any health relevance to the physiochemical paramaters? Several parameters exceed drinking water guidelines, although this is not for drinking water – maybe some of these levels should be of concern for public health?”
The authors acknowledge the comment made by the reviewer.
The manuscript has been revised taking into consideration the reviewer’s comments.
The Figure 5 was divided [Figure 5 and 6] and the followed table was renumbered.
Based on the chemical composition of thermal waters, there is an association between their physicochemical characteristics to therapeutic indications. This information was added [line 83-90].
It is true, although the main purpose of these waters is not for drinking, their physicochemical and microbiological composition should be analyzed carefully. For instance, according to the European legislation, “at source and during its marketing, a natural mineral water shall be free from: (a) parasites and pathogenic micro-organisms; (b) Escherichia coli and other coliforms and faecal streptococci in any 250 ml sample examined; (c) sporulated sulphite-reducing anaerobes in any 50 ml sample examined;
(d) Pseudomonas aeruginosa in any 250 ml sample examined.” Future studies are necessary to know better these waters, safeguarding public health.
“Title: The title is not grammatically correct for standard English. Alternative titles: “Does the hydgrogenome suggest that thermal contain bacteria beneficial for health?” “Are there benefits from thermal bacteria for health? Lessons from the hydrogenome””
The title was revised [line 3-4].
“Line 29: characterize (spelling)”
The word was revised [line 31].
“Line 32: replace “to” with “for”, insert “to” before analyse.”
This sentence was rewritten [line 34-36].
“Line 36-38: reword lines”
The sentence was rewritten [line 39-43].
“Line 39: communities is possessive rather than plural, so community’s”
It was changed [line 43].
“Line 42: “allowing the safe use” is leading reader to believe that all these waters are presumably safe. An alternative writing would provide greater margin for risk from harmful microbes and harmful chemicals that may be present that were not captured by these brief snapshots. Rather than “allowing the safe use”, it may be “increasing the likelihood of safe use””
This sentence was revised [line 46].
“Line 57: “knew” is leading statement. Better verb choice maybe “surmised””
The verb was changed [line 62].
“Line 66: What reference supports that therapeutic purpose of each water is linked to microbial diversity rather than minerals? Need to maybe mention more clearly in this statement that the mineral composition is influenced by the microbial diversity.”
As there are few studies that analyze the physiochemical characteristics together with the microbial communities existents of these water, for instance, Paduano et al. (2018) reported that the anti-rheumatic properties of sulphurous-bromine-iodine thermal water have associated with the presence of the followed bacteria belonging to genera Geothermobacterium, Thermus, Syntrophomonas Desulfomonile, Thiofaba and Thermodesulfovibrio, as described above in the manuscript [line 108-111].
“Line 100: Compresses or ‘comprises’”
*Stopped at line 100 with English editing.
The change was made [line 121].
“The overall NGS results and related information appears appropriate content for Water. The scientific method used was primarily exploratory in nature with no specific hypotheses.”
The authors acknowledge the comment made by the reviewer.
Round 2
Reviewer 1 Report
The authors improved the manuscript according to my suggestions and explained everything clearly in the answer, so my decision is acceptance of the manuscript in this form.
My comment was:
“In lines 122-123 you stated the number and richness of thermal waters in Spain and that some of them are used for therapeutic purposes. In the paper, clearly state why you chose three waters from Spain and two from Portugal. Which was the guiding thread? Why these waters?”
Response:
As mentioned in the manuscript, both chosen regions are made up of numerous hot springs. In addition, this work is the result of a partnership between a Portuguese University and a Spanish University. Considering these factors, the geographic proximity of these hot springs to the institutions and their characteristics, five of these waters were selected.
So my question was directed why did you choose exactly these 3 and not for example 10 of them.
Author Response
Reviewers #1:
Comments and Suggestions for Authors:
“The authors improved the manuscript according to my suggestions and explained everything clearly in the answer, so my decision is acceptance of the manuscript in this form.”
The authors acknowledge the comment made by the reviewer.
The manuscript has been revised taking into consideration the reviewer’s comments.
My comment was:
“In lines 122-123 you stated the number and richness of thermal waters in Spain and that some of them are used for therapeutic purposes. In the paper, clearly state why you chose three waters from Spain and two from Portugal. Which was the guiding thread? Why these waters?”
Response:
As mentioned in the manuscript, both chosen regions are made up of numerous hot springs. In addition, this work is the result of a partnership between a Portuguese University and a Spanish University. Considering these factors, the geographic proximity of these hot springs to the institutions and their characteristics, five of these waters were selected.
So my question was directed why did you choose exactly these 3 and not for example 10 of them.”
We agree that how many samples collected and analyzed more representativeness this study will have of both regions and municipalities agreement. However, this work was performed with two students with grants to obtain a master’s degree. For this chosen number of collected samples, the key factors that contributed were Iberian partnership, geographic proximity, time duration and associated cost. In this line, no more samples were selected. In addition, we also tried to choose natural thermal mineral waters with different emergency temperatures.
Reviewer 2 Report
The manuscript presented is an improvement; however, there are several moderate areas whereby revision or reconsideration of writing is needed. There are no major areas of concern. The original review indicated that extensive editing related to the English language was needed. It appears that this did not occur; however, the manuscript is a good contribution not to be hindered by many minor English edits; therefore, a thorough list has been provided for minor editing consideration.
MODERATE CONSIDERATIONS:
Section 2.3. What instrumentation or method(s) were used? Were they standard methods?
Line 296-298: The manuscript states that sulfur-rich water provides multiple health benefits. These should be supported by references
Line 312: GC content of the Table 3 should be explained with a footnote for GC indicating Guanine-Cytosine. Also, an explanation in the article describing the utility of the % of GC content measurement could be helpful as the levels observed in this study are within the range desired for metagenomic studies (Browne et al. 2020) https://academic.oup.com/gigascience/article/9/2/giaa008/5735313
Line 340: The term wealth index is not one I’m familiar with. Species richness and the various types of richness indices exist (e.g. Shannon, Simpson, etc.) Unless the index was calculated, the term index should not be used. Instead, a “higher species richness” compared to group one would likely be more appropriate.
Line 349: wealth or richness? -- showing a lower degree of species richness or a lower degree of taxonomic richness compared to the other group
Krona graphs are still a bit hard to read the text; however, that is common in other manuscripts due to the technology. If they cannot enlarge text, that is okay by my review as the authors do explain the figures using text which is helpful.
Line 492: needs reworded. “Additionally, the usefulness of this work can contribute to studies evaluating safety” – ?? I’m not sure of the intent of this statement to suggest how to best improve.
MINOR REVISIONS:
Line 29: regions of Portugal
Line 29: These collected [insert space]
Line 31: remove ‘the’ – using Next Generation Sequencing
Line 35: replace “of” with “within” to read “clustering within the Spanish waters…”
Line 60: insert “and”, use present verb tense. “wholesome and originating from an…
Line 77: associated “with” a specific
Line 117: insert “including”, thermal waters, “including the raw material”
Line 117: insert “and”, “or balneotheraphy, and increasing the safety in the used water.”
Line 126: remove ‘the” to read as “using NGS and the molecular technique”
Line 130: remove “as previously mentioned” … “the Iberian Peninsula, namely in the region…”
Line 144: replace compresses with “comprises”
Line 147: insert period after occurrences. Insert “These waters”
Line 147: “and occurrences. These waters are mainly of meteoric…”
Line 203: reword, “All of the sequencing was…”
Line 209: “This tool allows for the reporting of a wide variety of…”
Line 210: “obtained, as well as for evaluating the GC content…”
Line 224: subheading has extra spaces “of natural’
Line 227: replace acid with “acidic”
Line 246: replace was with “were”
Line 313: reword, “For each analyzed water sample, the refraction curve was calculated through…”
Line 314: remove “that”
Line 342: smaller should be “a lesser amount of microbial diversity”
Line 362: remove “Since these”. Sentence should start as “Krona graphs represent an important…”
Line 364: insert “the” – were from “the” Bacteria realm…
Line 394: maybe an extra space after ‘such as’
Line 420: species/genus (Thioimonas thermosulfata) should be italicized
Line 421: genus, Polaromonas, should be italicized
Line 442: insert “are”, being these genera “are ” used or “being these genera are quite used…”
Line 453: Remove comma. “ideal growth usually occurring…”
Line 459: the genus name Aquifex should be italicized
Line 464: remove comma, use period. Start new sentence. “Members of the genus Nitrospira belong…”
Line 464: Nitrospira is a genus and should be italicized
Line 469: Water is plural, so verb is ‘were’. The waters ES_PR and ES_TI were high in sulfur compounds (SO3-2 and S-2) and CO3.
Line 470: start new sentence. Insert “for”. “For the sample ES_BUR, the variable…”
Line 471: start new sentence. “The sample PT_SA was..”
Line 472: Check verb choice/spelling “constituied”
Line 482: replace contributed with “contributes”
Line 484: porpuses should be replaced with “purposes”
Line 488: replace “occurs mainly due to “was able to occur mainly due to…”
Line 490: insert “leading to”, … has the possibility of leading to the discovery of…
Line 493-Line 499 needs reworded and likely made into multiple sentences.
Author Response
Reviewers #2:
Comments and Suggestions for Authors:
“The manuscript presented is an improvement; however, there are several moderate areas whereby revision or reconsideration of writing is needed. There are no major areas of concern. The original review indicated that extensive editing related to the English language was needed. It appears that this did not occur; however, the manuscript is a good contribution not to be hindered by many minor English edits; therefore, a thorough list has been provided for minor editing consideration.”
The authors acknowledge the comment made by the reviewer.
The manuscript has been revised taking into consideration the reviewer’s comments.
Moderate considerations:
“Section 2.3. What instrumentation or method(s) were used? Were they standard methods?”
The physicochemical parameters were performed using standard methods. This information was added [line 185].
“Line 296-298: The manuscript states that sulfur-rich water provides multiple health benefits. These should be supported by references”
References were added [line 324].
“Line 312: GC content of the Table 3 should be explained with a footnote for GC indicating Guanine-Cytosine. Also, an explanation in the article describing the utility of the % of GC content measurement could be helpful as the levels observed in this study are within the range desired for metagenomic studies (Browne et al. 2020) https://academic.oup.com/gigascience/article/9/2/giaa008/5735313”
The information was added in Table 3 and in the text [Table 3, line 222-223, 337-341].
“Line 340: The term wealth index is not one I’m familiar with. Species richness and the various types of richness indices exist (e.g. Shannon, Simpson, etc.) Unless the index was calculated, the term index should not be used. Instead, a “higher species richness” compared to group one would likely be more appropriate.”
The sentence was rewritten [line 374].
“Line 349: wealth or richness? -- showing a lower degree of species richness or a lower degree of taxonomic richness compared to the other group”
The sentence was revised [line 383-384].
“Krona graphs are still a bit hard to read the text; however, that is common in other manuscripts due to the technology. If they cannot enlarge text, that is okay by my review as the authors do explain the figures using text which is helpful.”
The authors acknowledge the comment made by the reviewer.
Line 492: needs reworded. “Additionally, the usefulness of this work can contribute to studies evaluating safety” – ?? I’m not sure of the intent of this statement to suggest how to best improve.”
The sentence was rewritten [line 536-537].
Minor revisions:
“Line 29: regions of Portugal”
The sentence was revised [line 31].
“Line 29: These collected [insert space]”
The space was added [line 31].
“Line 31: remove ‘the’ – using Next Generation Sequencing”
This word was removed [line 33].
“Line 35: replace “of” with “within” to read “clustering within the Spanish waters…””
The sentence was revised [line 37].
“Line 60: insert “and”, use present verb tense. “wholesome and originating from an…”
The word was added [line 63].
“Line 77: associated “with” a specific”
The word was included [line 80].
“Line 117: insert “including”, thermal waters, “including the raw material””
The sentence was revised [line 120].
“Line 117: insert “and”, “or balneotheraphy, and increasing the safety in the used water.””
The sentence was revised [line 120].
“Line 126: remove ‘the” to read as “using NGS and the molecular technique””
The word was removed [line 129].
“Line 130: remove “as previously mentioned” … “the Iberian Peninsula, namely in the region…””
The sentence was revised [line 133-134].
“Line 144: replace compresses with “comprises””
The word was changed [line 149].
“Line 147: insert period after occurrences. Insert “These waters””
“Line 147: “and occurrences. These waters are mainly of meteoric…””
The sentence was revised [line 152].
“Line 203: reword, “All of the sequencing was…””
The sentence was revised [line 213].
“Line 209: “This tool allows for the reporting of a wide variety of…””
The sentence was rewritten [line 221].
“Line 210: “obtained, as well as for evaluating the GC content…””
The sentence was rewritten [line 221-223].
“Line 224: subheading has extra spaces “of natural’”
The extra space was removed [line 235].
“Line 227: replace acid with “acidic””
The word was changed [line 238].
“Line 246: replace was with “were””
The word was changed [line 257].
“Line 313: reword, “For each analyzed water sample, the refraction curve was calculated through…””
The sentence was rewritten [line 345].
“Line 314: remove “that””
The word was removed [line 346].
“Line 342: smaller should be “a lesser amount of microbial diversity””
The sentence was revised [line 377].
“Line 362: remove “Since these”. Sentence should start as “Krona graphs represent an important…””
The sentence was changed [line 399].
“Line 364: insert “the” – were from “the” Bacteria realm…”
It was added [line 402].
“Line 394: maybe an extra space after ‘such as’”
The extra space was removed [line 433].
“Line 420: species/genus (Thioimonas thermosulfata) should be italicized”
These words were italicized [line 461].
“Line 421: genus, Polaromonas, should be italicized”
This word was italicized [line 462].
“Line 442: insert “are”, being these genera “are ” used or “being these genera are quite used…””
This sentence was revised [line 484].
“Line 453: Remove comma. “ideal growth usually occurring…””
The comma was removed [line 495].
“Line 459: the genus name Aquifex should be italicized”
This word was italicized [line 501].
“Line 464: remove comma, use period. Start new sentence. “Members of the genus Nitrospira belong…””
These changes were performed [line 507].
“Line 464: Nitrospira is a genus and should be italicized”
This word was italicized [line 507].
“Line 469: Water is plural, so verb is ‘were’. The waters ES_PR and ES_TI were high in sulfur compounds (SO3-2 and S-2) and CO3.”
The sentence was changed [line 512.
“Line 470: start new sentence. Insert “for”. “For the sample ES_BUR, the variable…””
The sentence was revised [line 513].
“Line 471: start new sentence. “The sample PT_SA was..””
The sentence was revised [line 514].
“Line 472: Check verb choice/spelling “constituied””
The sentence was revised [line 515].
“Line 482: replace contributed with “contributes””
The word was replaced [line 525].
“Line 484: porpuses should be replaced with “purposes””
The word was replaced [line 527].
“Line 488: replace “occurs mainly due to “was able to occur mainly due to…””
The sentence was rewritten [line 531-532].
“Line 490: insert “leading to”, … has the possibility of leading to the discovery of…”
The sentence was revised [line 534].
“Line 493-Line 499 needs reworded and likely made into multiple sentences.”
This sentence was divided and revised [line 536-545].
Round 3
Reviewer 2 Report
The authors satisfactorily addressed nearly all concerns, and possibly sufficient for your decision to warrant publication. For the aspects about metal/mineral analysis, it's fairly common to report if analytical methods were used and by which instruments as there are some standard methods that can be accomplished using a variety of instruments. I suppose that since the authors describe the laboratory from whereby the analyses occurred, if there was a concerned reader, they could contact the authors. Overall, the authors addressed concerns.
