Effects of Chlorella vulgaris on phosphorus release from ferric phosphate sediment by consecutive cultivations

Iron phosphate (Fe–P) is a main phosphorus storage form, especially in phosphorus-polluted environments. The re-release of Fe–P is a problematic result during microalgal remediation. In this study, pre-incubated Chlorella vulgaris was cultured in a BG-11 culture medium with different amounts of Fe–P. The effects of Fe–P re-release on biomass, flocculation and removal of PO43− were investigated. The results indicated that C. vulgaris can promote the dissolution and release of Fe–P when the pH is 7, and the amount of Fe–P (ΔQ) released in 200 ml water reaches 0.055–0.45 mg d−1 under a C. vulgaris concentration of 5.6 × 105–8 × 105 cells ml−1. The growth of C. vulgaris was inhibited because of the flocculation behaviour of Fe3+ in the release stage, which is associated with a specific growth rate of 0.3–0.4 d−1 and a phosphorus removal rate below 30%. However, this process, in the long term, indicates a favourable transformation in which Fe–P becomes bioavailable under the action of C. vulgaris. Microalgae outbreaks may be triggered by persistent interactions between Fe–P and C. vulgaris. This study provides an important reference for the application of C. vulgaris in a Fe–P-rich environment.

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All figures. The lines that authors use to distinct the different cycles cover the symbols and the error bars. You may correct this by using, for instance, a thinner line. A more detailed discussion should be added. Please add a performance comparison with other similar works for displaying the advantages of this work.
Reviewer: 2 Comments to the Author(s) I cannot well understand what the authors told to us, a terrible writing style. The authors need to reorganize the manuscript by a fluent manner. Did you want to study the precipitation of Fe-P or the effects of C. vulgaris on P release from Fe-P in sediment? P release to solution is not a desirable result, so what is the meaning of P removal by consecutive cultivation with C. vulgaris? I don't think the authors have told us a clear story. The authors are recommended to rewrite the manuscript, including the title the manuscript, which is also not clear. For example, "effects of ^^^ on biomass", which biomass? Pls check the grammar issues of the manuscript carefully.
1. The English grammar in this paper must be improved for the reconsideration of publication. The manuscript needs to be edited for grammar and syntax. 2. The introduction was not well organized, pls reorganize this section by a logical way. At present version, we don't understand the main novelty of the work. 3. Line 17. The full name of C. vulgaris should be given. 4. Line 38. phosphorous? Pls check the similar mistakes throughout the manuscript. 5. Line 42. Chlorella vulgaris (C. vulgaris)? 6. Line 50. phosphate should be phosphate (PO43-). 7. Line 59. biology? 8. Line 66. but three general theories? 9. Line 77. Re? 10. Line 120. As shown in Table 1, all the spiked Fe3+ was transformed to Fe-P? 11. Line 204. this stage can be regarded as the adsorption of C. vulgaris? 12. Line 230. "The amount of released Fe3+ is the only factor that affects the C. vulgaris growth", the algae only need Fe3+ to sustain their growth? 13. Line 323. "To investigate the effect of Fe-P release on P removal", removed from the solution or sediment? It is hard to understand the authors' opinions.

See Appendices A & B.
Decision letter (RSOS-202198.R1) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Mr Chen: Title: Effects of Chlorella vulgaris on P release from ferric phosphate sediment by consecutive cultivation Manuscript ID: RSOS-202198.R1 Thank you for your submission, your above manuscript has now been reviewed. The comments from reviewers are included at the bottom of this letter.
In view of the criticisms of the reviewers, the manuscript has been rejected in its current form. However, a new manuscript may be submitted which takes into consideration these comments.
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For example, the authors use the word 'sediment' to refer to iron phosphate. However, strictly speaking, iron phosphate is not a sediment, a sediment is a much more complex system than a single compound. The authors also use the word 'structure' instead of 'species' to refer to PO43-, which is incorrect. They use the verb 'to reduce' instead of 'to decrease' for a species that reduces (Fe3+), which makes the reading confusing, and so on.

RSOS-211391.R0
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
Manuscript entitled "Effects of Chlorella vulgaris on P release from ferric phosphate sediment by consecutive cultivation" dealt with the effects of Fe-P re-release on biomass, flocculation and removal of PO43-by Chlorella vulgaris. The work is interesting and I would like to recommend its publication after some revisions. The word "sediment" is missing from the title in the manuscript. Please increase figures resolution. For instance, in figure 3 characters are difficult to read.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The authors need to provide a point-to-point response file to us.

Decision letter (RSOS-211391.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Mr Chen: Title: Effects of Chlorella vulgaris on P release from ferric phosphate sediment by consecutive cultivation Manuscript ID: RSOS-211391 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The editor assigned to your paper has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 30-Oct-2021. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance.We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor Comments to the Author: Dear authors, We would like you to revise your paper (point by point) in accordance with the referee and Subject Editor suggestions made to your previous manuscript (RSOS-202198), which can be found below (not including confidential reports to the Editor).

RSC Associate Editor:
Comments to the Author: The research presented in this draft is original and of interest to RSOS audience, however a professional proofreading service is highly recommended as the language needs a thorough revision in order to communicate your research clearly. For example, no sentences are provided in Section 2.2, which makes it difficult to understand it. Additionally, the quality of the figures must be improved, especially to avoid repetition. Please read carefully each of the comments from the reviewers and address each of them.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Manuscript entitled "Effects of ferric phosphate deposit on biomass and phosphorus removal by consecutive cultivation with Chlorella vulgaris" dealt with the effects of Fe-P re-release on biomass, flocculation and removal of PO43-by Chlorella vulgaris. The work is interesting and I would like to recommend its publication after some revisions. Page 4 line 41. "Therefore, C. vulgaris have been used in the treatment of P-contaminated water bodies, such as Chlorella vulgaris (C. vulgaris)" Please correct this sentence. Page 4 line 53. "PO43-is often combined with Fe3+ to form solid-phase compound (Ferric phosphate, Fe-P) that precipitate or adsorb to river sediments with the pollution expands on the timescale. The sentence is not clear, please correct. Please increase Figure 3 resolution. Characters are difficult to read. All figures. The lines that authors use to distinct the different cycles cover the symbols and the error bars. You may correct this by using, for instance, a thinner line. A more detailed discussion should be added. Please add a performance comparison with other similar works for displaying the advantages of this work.
Reviewer: 2 Comments to the Author(s) I cannot well understand what the authors told to us, a terrible writing style. The authors need to reorganize the manuscript by a fluent manner. Did you want to study the precipitation of Fe-P or the effects of C. vulgaris on P release from Fe-P in sediment? P release to solution is not a desirable result, so what is the meaning of P removal by consecutive cultivation with C. vulgaris? I don't think the authors have told us a clear story. The authors are recommended to rewrite the manuscript, including the title the manuscript, which is also not clear. For example, "effects of ^^^ on biomass", which biomass? Pls check the grammar issues of the manuscript carefully.
1. The English grammar in this paper must be improved for the reconsideration of publication. The manuscript needs to be edited for grammar and syntax. 2. The introduction was not well organized, pls reorganize this section by a logical way. At present version, we don't understand the main novelty of the work. 3. Line 17. The full name of C. vulgaris should be given. 4. Line 38. phosphorous? Pls check the similar mistakes throughout the manuscript. 5. Line 42. Chlorella vulgaris (C. vulgaris)? 6. Line 50. phosphate should be phosphate (PO43-). 7. Line 59. biology? 8. Line 66. but three general theories? 9. Line 77. Re? 10. Line 120. As shown in Table 1, all the spiked Fe3+ was transformed to Fe-P? 11. Line 204. this stage can be regarded as the adsorption of C. vulgaris? 12. Line 230. "The amount of released Fe3+ is the only factor that affects the C. vulgaris growth", the algae only need Fe3+ to sustain their growth? 13. Line 323. "To investigate the effect of Fe-P release on P removal", removed from the solution or sediment? It is hard to understand the authors' opinions.

RSOS-211391.R1
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Comments to the Author(s)
The authors performed the requested changes and corrections. The paper is now suitable for publication.

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
Two question, 1) The authors claim that they have polished the grammar, but I still find lots of sloppy English. For example, "The metabolites of C. vulgaris during the growth process can promote an increase in pH"? "in" should be "of". Pls improve the manuscript carefully again. 2) In the first round of review, I recommend the authors to correct the mistakes of the use of "full names" and (or) "abbreviated names", which don't mean that you need to give the full name of abbreviated name in the bracket following their abbreviated name or full name. For the 1st use of a Latin name of certain organism, you should use the full name, while for the 2nd use you can use the abbreviated name.

Decision letter (RSOS-211391.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Mr Chen:
Title: Effects of Chlorella vulgaris on P release from ferric phosphate sediment by consecutive cultivation Manuscript ID: RSOS-211391.R1 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The editor assigned to your paper has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 12-Jan-2022. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor Comments to the Author: In view of the criticisms of a reviewer, found at the bottom of this letter, two major issues still need to be addressed. Please read carefully each of the comments from the reviewer carefully and address each of them. As suggested previously, a professional proofreading service is highly recommended to improve the language.

RSC Subject Editor
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 2 Comments to the Author(s) Two question, 1) The authors claim that they have polished the grammar, but I still find lots of sloppy English. For example, "The metabolites of C. vulgaris during the growth process can promote an increase in pH"? "in" should be "of". Pls improve the manuscript carefully again. 2) In the first round of review, I recommend the authors to correct the mistakes of the use of "full names" and (or) "abbreviated names", which don't mean that you need to give the full name of abbreviated name in the bracket following their abbreviated name or full name. For the 1st use of a Latin name of certain organism, you should use the full name, while for the 2nd use you can use the abbreviated name.
Reviewer: 1 Comments to the Author(s) The authors performed the requested changes and corrections. The paper is now suitable for publication.

Decision letter (RSOS-211391.R2)
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Dear Mr Chen:
Title: Effects of Chlorella vulgaris on P release from ferric phosphate sediment by consecutive cultivation Manuscript ID: RSOS-211391.R2 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal. large amounts of phosphorus (P) that are discharged into river water significantly promote the eutrophication of water bodies, which not only damages the ecosystems of the river but also harms aquatic plants and animals [1,2]. Chlorella vulgaris (C. vulgaris) propagate by using inorganic nutrients, including nitrogen (N) and phosphorus (P), which means it have a high potential for removing inorganic nutrients from surrounding waters. Therefore, C. vulgaris is used in the treatment of eutrophic water bodies [3]. From the perspective of biomaterials, C.
vulgaris shows a high metal binding capacity because of the presence of polysaccharides and various functional groups of proteins or lipids on the cell wall surface (such as amino, hydroxyl, carboxyl, and sulfate) that can serve as binding sites for metal ions [4]. Currently, C. vulgaris has been widely used in wastewater (domestic sewage, industrial wastewater, and municipal wastewater) treatment based on its optimal adsorption or absorption capacity for N, P and metal cations (such as Fe 3+ , Cr 5+ , Ni 2+ , Zn 2+ , Cu 2+ , and Pb 2+ ) during its proliferation process [5][6][7][8][9][10]. P in river and lake is mainly present as PO4 3- [11], this structure can be readily combined with the metal cations (such as Fe 3+ , Ca 2+ and Al 3+ ) to form a precipitate and stored in the sediment of the river [12]. Iron phosphate (Fe-P) deposits are often formed by the combination of PO4 3-and Fe 3+ . The precipitate has the most important effect on the overlying water because of persistent release and periodic outbreaks of microalgae [13]. As a common sediment in rivers and lakes, however, Fe-P is considered to be an active compound but has very high release rate [14]. The release of PO4 3-and cations from the sediment could cause persistent pollution of upper water by the actions of microorganisms or human disturbance, which has become a potential hazard to the water environment [15,16]. Zhu et al. initially studied the internal nutrient release responds to algal bloom decay from Taihu Lake in China, the research shows sediments from the river mouth released P to the overlying water in the presence of blooms [17].
For now, the mechanism of Fe-P release under the action of C. vulgaris has not been clarified. There are several mechanisms that may be used to explain the release of Fe-P. The first is that the reductive dissolution is caused under the anoxic conditions of sediment-water interface by the respiration of C. vulgaris. Second, the competitive behavior of OHand PO4 3may be taken as the second part. Third, the reverse reaction of Fe 3+ hydrolysis is enhanced by a weak acid environment [18,19].
Release of excess Fe 3+ from Fe-P sediment can inhibit the growth of C. vulgaris and even exhibits toxicity effect, which is unfavorable for treatment of eutrophic wastewater [22,23].
Periodical algal bloom is induced by the release of P from the P-containing sediment [24,25]. In addition, Fe 3+ has a strong flocculation effect on C. vulgaris, because the surface of C. vulgaris is negatively charged and evenly suspended in liquid [20,21]. Therefore, chain reaction involving biomass production, pH fluctuation, flocculation, co-precipitation, and removal of PO4 3-can be triggered through the release dynamics of Fe-P sediment. However, these processes have not been employed to evaluate the advantages and disadvantages of C. vulgaris.
In practical applications, phosphorus contaminated water-body is repaired by C. vulgaris in an Fe-P rich environment. To simulate actual environmental conditions, in this study, using different amounts of FeCl3 and high content K2HPO4 to quantify Fe-P flocculated sediment, the sediment was cultured in BG-11 medium (removed PO4 3-and ferric ammonium) with C.
vulgaris during a continuous three-cycle culture. Effects of Fe 3+ /PO4 3-release from Fe-P on biomass, PO4 3-removal and flocculation were investigated. Finally, this paper evaluates the environmental effects of C. vulgaris in the Fe-P rich sediment rivers.