Nutrients recycle and the growth of Scenedesmus obliquus in synthetic wastewater under different sodium carbonate concentrations

This study illustrated the growth of Scenedesmus obliquus and recycle of nutrients in wastewater combined with inorganic carbon under autotrophic conditions. Scenedesmus obliquus was cultivated under different conditions by adding sodium carbonate (Na2CO3) at 15–40 mg l−1 separately in wastewater containing high nitrogen and phosphorus content. The growth characteristics of S. obliquus, pH and dissolved inorganic carbon (DIC) changes of microalgae liquid, the recycle rate of ammonia and phosphorus and lipid content were determined. The changes of pH and DIC showed that S. obliquus could use Na2CO3 to grow, with lipid contents of 18–25%. Among all Na2CO3 concentrations, 20 mg l−1 was the optimum, of which S. obliquus had the highest NH3-N recycle of 52% and PO43–P recycle of 67%. By the 14th day, its biomass production also reaches the maximum of 0.21 g l−1. However, inorganic carbon fixation rate was inversely proportional to its concentration. Moreover, the biomass was in positive correlation with the Na2CO3 concentration except 20 mg l−1, which provided a possibility that S. obliquus could be acclimatized to adjust to high concentrations of inorganic carbon to promote biomass accumulation and recycle of nutrients.

culture was left in the flask. What is the effect of the above procedure on the culture ? Furthermore, did authors experience problems with water evaporation in the flasks ? Please discuss it. 7. Section 4.1. For the cultivation experiments an initial biomass concentration of around 60 mg/L was used. What is the corresponding cell number ? 8. Section 4.1. No lag phase was observed in all sodium carbonate concentrations used. Please explain it. 9. Page 5, line 1. Please provide a reference for the optimal C/N ratio. 10. Figure 4. The initial ammonia and phosphorus concentration does not correspond to the quantity added to the medium. 11. Section 4.4. The second paragraph should be removed, since the harvest process was not the aim of the study. 12. Section 4.5. What was the FAMe's composition in the present study ?

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

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

Comments to the Author(s)
In this manuscript, the authors investigated the nutrients recycle and the growth of Scenedesmus obliquus in wastewater under different sodium carbonate concentrations. The topic is interesting, and overall, the methods and the results were presented satisfactorily. However, some parts of the manuscript were not presented well, and revisions are needed prior to a possible publication in Royal Society Open Science. Detailed comments are listed as follows.
On Introduction 1.In the Introduction, the current state in the research field should be presented with more details, and the authors should describe more recent progresses on the response of microalgae and plants to pollutants, for example, data and results described in the following articles: Responses of microalgae Coelastrella sp. to stress of cupric ions in treatment of anaerobically digested swine wastewater, Bioresource Technology, 2018, 251: 274-279; Effects of copper ions on removal of nutrients from swine wastewater and release of dissolved organic matters in duckweed systems, Water Research, 2019, 158: 171-181.
On Material and Methods 2.3.2. Preparation of synthetic wastewater: The authors should set up a control group, and indicate the initial concentration of NH3-N and PO43-P in synthetic wastewater.
3. The author could examine and discuss more on the content of enzymes or other physiological and biochemical properties to reflect the effects of Na2CO3 on microalgae growth either in this section or later.
On Results and Discussion 4. Error bars should be provided in the figures whenever available, otherwise, the authors should explain the reasons and effect on data credibility. 5.4.2 Absorption and utilization of Na2CO3 by phytosynthesis of microalgae: Why the carbon sequestration of photosynthesis could increase the pH? 6.4.3. Recycle of ammonia and phosphorus at different Na2CO3 concentrations: The concentration of ammonia in the solution was still at 80 mg L-1 after 6 days, why the authors said that the removal efficiency of ammonia was restricted by the exhaustion of nutrients? At the end of this work, whether the ammonia concentrations are meeting the emission standards? 7.4.3. Recycle of ammonia and phosphorus at different Na2CO3 concentrations: Besides microalgae, many aquatic plants and microorganisms are also used for the nutrient removal from the high nitrogen and phosphorus wastewater, such as duckweed and bacteria. What is the difference between microalgae and their removal efficiency? The following literature and the article mentioned above could serve this purpose on some aspects: Phytoremediation of anaerobically The editor assigned to your manuscript 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 your revised paper before 25-Sep-2019. 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.
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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: (There are no comments.) RSC Subject Editor: Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) This paper is concerned to determine a potential contribution of Scenedesmus obliquus and recycle of nutrients in wastewater combined with inorganic carbon under autotrophic conditions. However, the manuscript is purely descriptive analytical protocols does not report on significant advances in scientific research. I have considered it does not bring about sufficient information to justify publication. For all these reasons I do not recommend its publication in Environmental Technology in the present state.

Reviewer: 2
Comments to the Author(s) General Comments This manuscript investigates the role of different concentrations of Na2CO3 as inorganic carbon (IC) source for the growth of Scenedesmus obliquus. The paper is potentially interesting for the readers of the Journal and could be considered for publication after changes.
Specific Comments 1. Title of the paper. Since the substrate used was BG-11 supplemented with nutrients the term "synthetic wastewater" should be used instead of wastewater. 2. Section 3.1. Please check the composition of BG-11 medium. It seems that NaNO3 and ferric ammonium citrate are different from the typical BG-11 composition. If this the case, the BG-11 medium should be mentioned as modified BG-11. 3. Section 3.1. Describe the illumination system. 4. Section 3.2. Please describe how many replicates of every sodium carbonate concentration were conducted. A standard deviation should be provided in the figures. 5. Section 3.2. Please clarify that the final concentration of sodium carbonate was between 15 and 40 mg/L. 6. Section 3.3. The authors mentioned that a 10-mL sample volume was withdrawn each sampling day. Based on the above, it seems that at the end of the experiment less than the half-volume of culture was left in the flask. What is the effect of the above procedure on the culture ? Furthermore, did authors experience problems with water evaporation in the flasks ? Please discuss it. 7. Section 4.1. For the cultivation experiments an initial biomass concentration of around 60 mg/L was used. What is the corresponding cell number ? 8. Section 4.1. No lag phase was observed in all sodium carbonate concentrations used. Please explain it. 9. Page 5, line 1. Please provide a reference for the optimal C/N ratio. 10. Figure 4. The initial ammonia and phosphorus concentration does not correspond to the quantity added to the medium. 11. Section 4.4. The second paragraph should be removed, since the harvest process was not the aim of the study. 12. Section 4.5. What was the FAMe's composition in the present study ?
Reviewer: 3 Comments to the Author(s) In this manuscript, the authors investigated the nutrients recycle and the growth of Scenedesmus obliquus in wastewater under different sodium carbonate concentrations. The topic is interesting, and overall, the methods and the results were presented satisfactorily. However, some parts of the manuscript were not presented well, and revisions are needed prior to a possible publication in Royal Society Open Science. Detailed comments are listed as follows.
On Introduction 1.In the Introduction, the current state in the research field should be presented with more details, and the authors should describe more recent progresses on the response of microalgae and plants to pollutants, for example, data and results described in the following articles: 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 28-Nov-2019. 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.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.

Dear Editors and reviewers:
Thank you for your letter dated 02 September. We were pleased to know that our work was rated as potentially acceptable for publication in journal of Royal Society Open Science. We appreciate the reviewers for the time and effort that they have put into reviewing the previous version of the manuscript. Their suggestions have enabled us to improve our work. Based on the instructions provided in your letter, we would like to resubmit the manuscript (RSOS-191214) entitled "Nutrients recycle and the growth of Scenedesmus obliquus in wastewater under different sodium carbonate concentrations", with all the changes highlighted by using different color (red). There are no conflicts of interest in manuscript and all authors are in agreement with the content of the manuscript. This work is not under active consideration for publication elsewhere, has not been accepted for publication, nor has it been published.
Point-by-point responses to the reviewers' comments are enclosed after the letter for your consideration.
We deeply appreciate your consideration of our resubmitted manuscript and we look forward to receiving comments from the reviewers. Comments to the Author(s) This paper is concerned to determine a potential contribution of Scenedesmus obliquus and recycle of nutrients in wastewater combined with inorganic carbon under autotrophic conditions. However, the manuscript is purely descriptive analytical protocols does not report on significant advances in scientific research. I have considered it does not bring about sufficient information to justify publication. For all these reasons I do not recommend its publication in Environmental Technology in the present state.

Reviewer: 2
Comments to the Author(s) General Comments This manuscript investigates the role of different concentrations of Na2CO3 as inorganic carbon (IC) source for the growth of Scenedesmus obliquus. The paper is potentially interesting for the readers of the Journal and could be considered for publication after changes.
Specific Comments 1. Title of the paper. Since the substrate used was BG-11 supplemented with nutrients the term "synthetic wastewater" should be used instead of wastewater.
Author's responses: We modified the title to "Nutrients recycle and the growth of Scenedesmus obliquus in synthetic wastewater under different sodium carbonate concentrations" according to the comment (Page 1).