the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 01 Oct 2025
Technical note: Development of an extraction protocol and colorimetric analysis for alginate in marine sediment
Abstract. Transportation of organic carbon from coastal macroalgae, particularly brown algae, to deeper ocean layers has recently attracted attention as an effective mechanism for carbon sequestration. However, no observational data are currently available on the amounts of organic carbon derived from brown algae in marine environments such as seawater or sediments. In this study, we developed an extraction protocol for alginate, a polysaccharide unique to brown algae that accounts for 20–30 % of their dry weight, to provide quantitative and direct evidence of brown algal carbon sequestered in ocean sediments. Alginate extraction and colorimetric analysis methods are well established in food chemistry: we modified these techniques to apply them to marine sediments, which are characterized by high concentrations of cations (e.g., Ca, Mg, Fe) and humic substance-like high-molecular-weight organic compounds. We applied this new method to sediment samples collected from coastal waters around Hokkaido, Japan. Alginate contents were quantified as 6.11–26.0 mg m−2 in Funka Bay, 39.0–41.3 mg m−2 in Hakodate Bay, 11.8–14.7 mg m−2 off Cape Esan, and 58.3–74.1 mg m−2 off Muroran.
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Notice on discussion status
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
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Preprint
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Supplement
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The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
- Preprint
(1131 KB) - Metadata XML
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Supplement
(594 KB) - BibTeX
- EndNote
- Final revised paper
Journal article(s) based on this preprint
Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2025-4577', Anonymous Referee #1, 01 Dec 2025
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AC1: 'Reply on RC1', Sota Nakazato, 22 Dec 2025
We sincerely appreciate the time you devoted to reviewing our manuscript and for providing constructive and insightful comments. We are also grateful for your positive evaluation of the protocol from both scientific and practical perspectives. Based on your valuable suggestions, we have prepared detailed responses to each of your comments below. We hope that our explanations adequately address your concerns, and we thank you again for your thoughtful feedback.
1: Unify the format of units, w/w or w·w−1
To address this comment, we have unified the formatting of concentration units throughout the manuscript. Specifically, all solution concentrations previously expressed as “% (w/v or v/v)” have been revised to “g L−1” or “mL L−1,” as appropriate. We have also replaced the descriptive ratio-style expression for alginate in sediments, “mg per 300 g (alginate/wet weight of sediment),” with the standardized notation “mg 300 g−1”.
2: Why yield is expressed per 300 g ?
We originally expressed alginate yield as “mg per 300 g” to indicate the amount of alginate extracted from each sediment sample, whose wet weight was fixed at 300 g. In response to your comment, we have standardized this notation to “mg 300 g−1” throughout the manuscript.
The rationale for selecting 300 g as the sediment sample weight has also been clarified in Section 2.3, where we added the statement: “The weight of 300 g corresponds approximately to the amount of sediment collected from the upper 0–1 cm layer using a grab sampler.”
To further improve clarity, we have added a note indicating “Sediment sample weight = 300 g (wet)” in Tables 2, 5, and 8, where alginate yield (mg 300 g−1) is reported. These revisions improve consistency and readability in the presentation of the alginate yield data.
3: Latin names in References should be in italics
Thank you for pointing this out. All Latin names appearing in the references have been converted to italic font in accordance with the journal’s formatting requirements.
Please note that these changes will be incorporated into the revised manuscript.
Citation: https://doi.org/10.5194/egusphere-2025-4577-AC1
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AC1: 'Reply on RC1', Sota Nakazato, 22 Dec 2025
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RC2: 'Comment on egusphere-2025-4577', Anonymous Referee #2, 24 Mar 2026
This is a useful paper, its main contribution is methodological. The authors show that the original food-chemistry protocol performs poorly in sediment, and that their modified protocol improves recovery substantially. That is the strongest result in the paper, and it should be the main message.
My main concern is overstatement. The paper often sounds as if it has cleanly measured brown-algal carbon in sediment. But the evidence presented is more limited. The method measures uronic acids. The authors themselves show some remaining non-alginate signal, and the enzyme experiment only supports that at least part of the measured material is alginate. The paper is much stronger if framed as a promising sediment method with a first field application, not as definitive proof of brown-algal carbon sequestration.
Main comments
The paper should center its novelty more clearly. The real novelty is not the broad blue-carbon context. It is that the authors adapted an existing alginate method to a much harder matrix, marine sediment, and identified two practical changes that matter: adding EDTA and removing ethanol precipitation.
The claims about specificity should be softened. The phytoplankton test still gives a residual signal equivalent to 23% of the sediment signal, which is not trivial. The enzyme test also leaves a large residual signal, and the authors conclude only that alginate accounts for at least one-third of the uronic acids detected. That supports the presence of alginate, but not a clean one-to-one conversion from color signal to alginate alone. The wording should reflect that more carefully throughout.
The conclusion goes a bit too far. The manuscript currently says the method provides “quantitative and direct evidence” of brown-algal carbon in ocean sediments, and later links the findings to coastal carbon sequestration. That feels stronger than the data support. The study shows alginate-like material in surface sediments. It does not directly show long-term burial or sequestration. I would recommend more cautious language here.
The introduction is longer than needed. It spends too much time on general uronic-acid chemistry before getting to the actual gap. I would shorten that part and move faster to the practical problem: no established method exists for alginate in marine sediment, where cations, low concentrations, and other uronic-acid sources complicate the analysis.
There is also a wording inconsistency that should be fixed. The abstract calls alginate “unique to brown algae,” but the introduction later states that alginate is also produced by some bacteria. The authors should use one careful formulation throughout.
The paper has a real contribution and the method improvement seems meaningful. The main issue is in the framing, not the core idea. With tighter wording, clearer explanations, and more cautious claims, the manuscript would become stronger and more convincing.
Citation: https://doi.org/10.5194/egusphere-2025-4577-RC2 -
AC2: 'Reply on RC2', Sota Nakazato, 01 Apr 2026
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2025/egusphere-2025-4577/egusphere-2025-4577-AC2-supplement.pdf
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AC2: 'Reply on RC2', Sota Nakazato, 01 Apr 2026
Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2025-4577', Anonymous Referee #1, 01 Dec 2025
The manuscripts presents a study on the development of a method adapted to the determination of alginate in sediments.
The interest of this modified protocol is well justified, both from a scientific and from practical approaches. The work planning is well presented and the results are clearly presented and discussed.The authors used the protocol to characterize samples from different locations.
Only minor comments are suggested:
Unify the format of units, w/w or w·w-1
Why yield is expressed per 300 g?
Latin names in References should be in italics
Citation: https://doi.org/10.5194/egusphere-2025-4577-RC1 -
AC1: 'Reply on RC1', Sota Nakazato, 22 Dec 2025
We sincerely appreciate the time you devoted to reviewing our manuscript and for providing constructive and insightful comments. We are also grateful for your positive evaluation of the protocol from both scientific and practical perspectives. Based on your valuable suggestions, we have prepared detailed responses to each of your comments below. We hope that our explanations adequately address your concerns, and we thank you again for your thoughtful feedback.
1: Unify the format of units, w/w or w·w−1
To address this comment, we have unified the formatting of concentration units throughout the manuscript. Specifically, all solution concentrations previously expressed as “% (w/v or v/v)” have been revised to “g L−1” or “mL L−1,” as appropriate. We have also replaced the descriptive ratio-style expression for alginate in sediments, “mg per 300 g (alginate/wet weight of sediment),” with the standardized notation “mg 300 g−1”.
2: Why yield is expressed per 300 g ?
We originally expressed alginate yield as “mg per 300 g” to indicate the amount of alginate extracted from each sediment sample, whose wet weight was fixed at 300 g. In response to your comment, we have standardized this notation to “mg 300 g−1” throughout the manuscript.
The rationale for selecting 300 g as the sediment sample weight has also been clarified in Section 2.3, where we added the statement: “The weight of 300 g corresponds approximately to the amount of sediment collected from the upper 0–1 cm layer using a grab sampler.”
To further improve clarity, we have added a note indicating “Sediment sample weight = 300 g (wet)” in Tables 2, 5, and 8, where alginate yield (mg 300 g−1) is reported. These revisions improve consistency and readability in the presentation of the alginate yield data.
3: Latin names in References should be in italics
Thank you for pointing this out. All Latin names appearing in the references have been converted to italic font in accordance with the journal’s formatting requirements.
Please note that these changes will be incorporated into the revised manuscript.
Citation: https://doi.org/10.5194/egusphere-2025-4577-AC1
-
AC1: 'Reply on RC1', Sota Nakazato, 22 Dec 2025
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RC2: 'Comment on egusphere-2025-4577', Anonymous Referee #2, 24 Mar 2026
This is a useful paper, its main contribution is methodological. The authors show that the original food-chemistry protocol performs poorly in sediment, and that their modified protocol improves recovery substantially. That is the strongest result in the paper, and it should be the main message.
My main concern is overstatement. The paper often sounds as if it has cleanly measured brown-algal carbon in sediment. But the evidence presented is more limited. The method measures uronic acids. The authors themselves show some remaining non-alginate signal, and the enzyme experiment only supports that at least part of the measured material is alginate. The paper is much stronger if framed as a promising sediment method with a first field application, not as definitive proof of brown-algal carbon sequestration.
Main comments
The paper should center its novelty more clearly. The real novelty is not the broad blue-carbon context. It is that the authors adapted an existing alginate method to a much harder matrix, marine sediment, and identified two practical changes that matter: adding EDTA and removing ethanol precipitation.
The claims about specificity should be softened. The phytoplankton test still gives a residual signal equivalent to 23% of the sediment signal, which is not trivial. The enzyme test also leaves a large residual signal, and the authors conclude only that alginate accounts for at least one-third of the uronic acids detected. That supports the presence of alginate, but not a clean one-to-one conversion from color signal to alginate alone. The wording should reflect that more carefully throughout.
The conclusion goes a bit too far. The manuscript currently says the method provides “quantitative and direct evidence” of brown-algal carbon in ocean sediments, and later links the findings to coastal carbon sequestration. That feels stronger than the data support. The study shows alginate-like material in surface sediments. It does not directly show long-term burial or sequestration. I would recommend more cautious language here.
The introduction is longer than needed. It spends too much time on general uronic-acid chemistry before getting to the actual gap. I would shorten that part and move faster to the practical problem: no established method exists for alginate in marine sediment, where cations, low concentrations, and other uronic-acid sources complicate the analysis.
There is also a wording inconsistency that should be fixed. The abstract calls alginate “unique to brown algae,” but the introduction later states that alginate is also produced by some bacteria. The authors should use one careful formulation throughout.
The paper has a real contribution and the method improvement seems meaningful. The main issue is in the framing, not the core idea. With tighter wording, clearer explanations, and more cautious claims, the manuscript would become stronger and more convincing.
Citation: https://doi.org/10.5194/egusphere-2025-4577-RC2 -
AC2: 'Reply on RC2', Sota Nakazato, 01 Apr 2026
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2025/egusphere-2025-4577/egusphere-2025-4577-AC2-supplement.pdf
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AC2: 'Reply on RC2', Sota Nakazato, 01 Apr 2026
Peer review completion
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Sota Nakazato
Takashi Kawahara
Reiji Ichii
Shingo Akita
Akira Inoue
Masaki Fujita
Hideyuki Kurihara
Mitsuhiro Nakaya
Atsushi Ooki
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
- Preprint
(1131 KB) - Metadata XML
-
Supplement
(594 KB) - BibTeX
- EndNote
- Final revised paper
The manuscripts presents a study on the development of a method adapted to the determination of alginate in sediments.
The interest of this modified protocol is well justified, both from a scientific and from practical approaches. The work planning is well presented and the results are clearly presented and discussed.The authors used the protocol to characterize samples from different locations.
Only minor comments are suggested:
Unify the format of units, w/w or w·w-1
Why yield is expressed per 300 g?
Latin names in References should be in italics