PONE-D-22-18432Optimising a method for aragonite precipitation in simulated biogenic calcification mediaPLOS ONE

Dear Dr. Kellock,

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Additional Editor Comments:

All the reviewers recognize the merit of the manuscript and they recommend that it needs to be carefully revised before the acceptance. I suggest the author read the reviewer's comments (in the attachment) and revise it point-by-point accordingly. The highlight in the revised part are highly preferred for our review.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I think the goal of the paper is important and interesting to a specific group working on CaCO3 mineralization. However, I find it quite difficult to follow the paper. I think the paper can be restructured and wrote more concisely and logically. Also, some figures are too sloppy. For example,

1. The x axis in the Figure 3b seems wrong. Why two axes are showed here?

2. Figure 6 does not have alphabets. I guess I can figure it out myself, but that’s annoying. What’s the unit of precipitation rate here? The same as the one mentioned before? ( btw, I was very interested in the lag time measurement, but find it difficult to follow in the paper, also, when you reference to the figure, please be more precise. In many cases, you only refer to figure xx. It is better to go to a specific panel.

3. Figure 8a. obvious typos.

4. Figure 9, don’t use “left” and “right” for the figures.

Overall, I think the paper needs to be carefully/majorly revised.

Reviewer #2: In this study, the authors synthesized aragonite in simulated biogenic calcification solution using a titration system and investigated the influence of aspartic acid on the structure and morphology. They found that the seeded crystallization experiment was more reproducible than unseeded experiments and that the full width half maxima of the v1 peak in Raman increases with increasing aspartic acid concentration. Firstly, I do not gain much new knowledge regarding the preparation method of aragonite, as claimed by the title of the manuscript, although they show that by adding seeds in the solution the experiments are more reproducible. I suggest the authors to change the title to better reflect the content of the work, which should be focusing on the precipitation rate and the influence of aspartic acid. Secondly, it is well known that the incorporation of aspartic acid in calcite could results a change of the lattice parameter, and may change the FWHM of the Raman peaks. It is kind of interesting to see such a change in aragonite, suggesting either the crystallinity of aragonite decreases or aspartic acid is incorporated in the structure of aragonite. The authors should perform more chemical and structural characterization, such amino acid analysis, XRD, infrared, and provide some explanations. Therefore, I do not recommend publication of this manuscript in its current form.

Reviewer #3: The authors present a well written article with a systematic experimental approach involving the use of Raman spectroscopy. A reliable method to study the impact of various organic and inorganic components would be highly beneficial to better understand geochemistry and calcified marine life through various lifecycles. However, this article needs major revision or resubmission before further consideration for the following reasons.

1. The references do not match the statements in main text and there seems to be an extra reference somewhere. This makes it difficult to read the manuscript and understand what the manuscript is talking about or verify the validity of the statements. For example:

Line 87-91: Ref 16 does not use Raman spectroscopy nor discusses CaCO3, though the statement could be talking about reference 15.

Line 418 – Ref 35 uses no seawater in their study

Line 427 to 433, references don’t match the statement.

2. Line 87-91: Reference 3 discusses rotational disorder from Mg or other impurities cannot be easily detected from aragonite but can be detected from Mg-calcite, which argues against the authors use of Raman for detecting disorder as a result of Sr or organics in aragonite using raman.

3. Figure 2. I’m concerned about the use of the v1 peak as an identifier for the presence of aragonite because this peak seems to exists for all polymorphs of CaCO3. It could be useful to see a combined full spectrum for all the samples/measurements against a reference aragonite, calcite, ACC spectra etc , somewhere in the SI or repository, especially at 100 -350 cm-1- wavenumber which can better identify the polymorph than the v1 peak (Ref: https://www.nature.com/articles/s41467-018-07601-3 ). Because it is hard to believe that precipitates other than aragonite are not produced at such high supersaturations or by the ex situ method of analysis involving drying of the precipitates. Is it possible that presence of precipitates other than aragonite could cause small changes in that peak? Especially a broadening caused by the presence of amorphous calcium carbonate which have relatively more disorder around CO3 compared to aragonite, rather than the presence of polyaspartic acid as the authors suggests later on. ACC formation is a common intermediate when using highly charged polymers which form polymer-induced liquid precursor (PILP) droplets in presence of cations and anions. In the PILP method, the polymer is typically excluded from the crystal that forms from aggregates of ACC+polymer nanoparticles (Ref: https://www.nature.com/articles/s41467-018-05006-w ); this exclusion of the polymer also conflicts with the author’s interpretation that occlusion of aspartic acid in the aragonite causes peak broadening

4. Line 246 to 262. It is understandable that the precipitation is not reproducible without the presence of aragonite seeds, however, this section is confusing to me, especially relationship between the supersaturation, [DIC] and CO2. Why does the higher omega sample produce lower precipitation? One would expect it to be the other way around.

5. Line 483 – 503: Following point#3 above, is there any evidence to show that higher amorphous mineral is not formed with higher aspartic acid concentrations and results in the peak broadening? Aspartic acid is too large to cause lattice level disorder or incorporation into the lattice that was previously reported for Mg incorporation into calcite lattice.

6. Line 521: It would be helpful for the reader to have the conclusion clearly state how the article has optimized the precipitation. For example, “We optimized a method for the precipitation of synthetic aragonites under simulated biological conditions by using aragonite seeds as substrate to improve reproducibility of precipitation rates” or better

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Optimising a method for aragonite precipitation in simulated biogenic calcification media

PONE-D-22-18432R1

Dear Dr. Kellock,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Amitava Mukherjee, ME, Ph.D.

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have addressed all my concerns and I have no further comments. I suggest publication of the manuscript.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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