Review Reports - Effects of Nitrate Source (Artificial and Fishpond) and UV Radiation on Physiological, Photosynthetic, and Biochemical Parameters of <i>Porphyra dioica</i> for Sustainable Integrated Multitrophic Aquaculture (IMTA)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting paper with of work and analysis ; however I think we can improve the quality and make it better paper.

Introduction:

In order to show the importance of the subject you should give more data on the production of porphyra and its transformation both in Spain, Europe and worldwide. Same remark for IMTA system.

Did some work before you on the effect of nitrogen on porphyra? if yes, you should give the reference. If no, you should say it and this is will show the importance of the work. As researchers we are not alone, many other researchers are making studies and try to improve our knowledge.

Material and methods:

Too many analysis and many methods well described. However we have a problem with the samples: the biological material was sampled only one time in june 2024. Is there any variation if we consider another month or another year ?

the same remark for fish: why Chelon labrosus? date of sampling ? quantity? did you consider the variation during the year?

Results:

Off course many results since many of analysis were carried out. But we are surprised some times by some results: for example in Table 5, there is no significant impact of nitrogen application on % C, % N, % S in porphyra.

In table 2, you have too big standard deviation which normally make difficult to get significant results. I think you should give explain.

Discussion:

Finally i found in the discussion two references of other works. you compared with their results but I think you should try to find more references.

i wonder why these two concentrations of nitrogen were choosen and if you expect to continue the work with other concentrations especially when many parameters here were not affected by these concentrations. you can also as perspective to test other source of nitrogen from poultry for example.

Other remarks:

Tables in figure 2 too small

number of replicates is not offen given

Figure 3 : no SD given for PE:PC

Table 5: precise if it is % dry or fresh weight

Author Response

This is an interesting paper with of work and analysis ; however I think we can improve the quality and make it better paper.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Introduction:

In order to show the importance of the subject you should give more data on the production of Porphyra and its transformation both in Spain, Europe and worldwide. Same remark for IMTA system.

Response: We agree, and have improved this section.

Material and methods:

Response: We acknowledge the reviewer’s comment. Porphyra sp. exhibits clear seasonality, with the macroscopic gametophytic (blade) phase predominant in winter and the microscopic conchocelis phase in summer. In this study, we focused exclusively on the blade phase, the commercial phase, where temperature and photoperiod are expected to be similar across years. Our recent observations (manuscript in preparation) indicate that nutrient availability can vary depending on the region (e.g., Atlantic Ocean vs. Mediterranean Sea), potentially influencing the production of bioactive compounds. However, in the present work we used algae from the same site, period, and physiological stage, comparing bioactive compound production with or without fishpond effluents after an acclimation period under controlled laboratory conditions. The objective was to understand the specific effect of the fishpond effluents, keeping all other environmental and biological factors constant.

The same remark for fish: why Chelon labrosus? date of sampling ? quantity? did you consider the variation during the year?

Response: We acknowledge the reviewer’s question. Chelon labrosus (thick-lipped grey mullet) was selected because it is a common native species in local aquaculture systems (García Marquez et al., 2022) and integrated multitrophic aquaculture (IMTA) practices, widely used in coastal ponds in southern Spain. It is a low trophic species, mainly herbivorous, and its aquaculture is promoted by international organizations as FAO. We have investigated on aquaponic by using this species to produce strawberry and the fishes were feeding by microalgae (García Marquez et al., 2023; Korbee et al., 2025). Their effluents represent a realistic and regionally relevant nutrient source for Porphyra cultivation. Fishpond water was collected in June 2024, at the same time as the algal material, ensuring consistency between both biological sources. The volume sampled was sufficient to maintain the experimental treatments throughout the acclimation and testing periods. Seasonal variation in effluent composition (especially nitrogen forms and concentration) is expected during the year, depending on temperature, feeding rate, and pond management. However, our aim was to evaluate the specific physiological response of Porphyra under a representative effluent condition, not to describe seasonal pond dynamics. We have added the sampling period (June 2024) to clarify this point in the manuscript.

García-Márquez J , Cerezo IM, Figueroa, FL, Abdala-Díaz RT, Arijo S (2022). First evaluation of associated gut microbiota in wild thick-lipped grey mullets (Chelon labrosus, Risso 1827) Fishes 7, 209. https://doi.org/10.3390/fishes7040209

García-Márquez, J., Álvarez-Torres, D., Cerezo, I. M., Domínguez-Maqueda, M., Figueroa, F. L., Alarcón, F. J., ... & Arijo, S. (2023). Combined dietary administration of Chlorella fusca and ethanol-inactivated Vibrio proteolyticus modulates intestinal microbiota and gene expression in Chelon labrosus. Animals, 13(21), 3325.

Korbee N, Bautista B, García Sanchez M, Cobos O, Ferres-García JL, Figueroa FL, Medrano E (2025) Evaluating hydroponics and aquaponics: comparative insights into sustainavailability and strawberry quality . Agricultural water management 312:109412

Results:

Of course many results since many of analysis were carried out. But we are surprised some times by some results: for example in Table 5, there is no significant impact of nitrogen application on % C, % N, % S in Porphyra.

Response: We agree with the reviewer, and we have added a sentence in the discussion noting that ‘longer experimental durations might reveal more pronounced metabolic adjustments or delayed responses to nutrient and UVR variations’.

Another thing surprised me: you have big standard deviation and this is can be due to the number of repetitions, the precision of the method or to the person making the analysis. This is very evident in Figure 2 and in Table 2. In table 2, you have too big standard deviation which normally make difficult to get significant results. I think you should give explain.

Response: Thank you for the observation. The relatively high standard deviations observed in some parameters (Figure 2 and Table 2) likely reflect the intrinsic biological variability of P. dioica thalli and the short duration of the experiment (4 days), rather than methodological imprecision. All analyses were performed in triplicate using standardized protocols and the same calibrated instruments to minimize technical variation. However, since red macroalgae often exhibit heterogeneous photosynthetic responses across individual thalli, some dispersion in the results is expected. We have clarified this point in the discussion, noting that ‘It is important to note that the relatively high standard deviations observed in some photosynthetic parameters likely reflect natural variability among thalli. Longer experimental periods could reduce this variability by allowing seaweeds to acclimate more fully to the experimental conditions’.

Discussion:

Finally I found in the discussion two references of other works. you compared with their results but I think you should try to find more references.

Response: We agree, and have added more references in discussion section.

I wonder why these two concentrations of nitrogen were choosen and if you expect to continue the work with other concentrations especially when many parameters here were not affected by these concentrations. you can also as perspective to test other source of nitrogen from poultry for example.

Response: Thank you for your comment. The explanation for the 5 mM and 3 mM nitrate concentrations has been clarified in the Materials and Methods section ‘The 5 mM NO₃⁻ concentration was based on previous studies indicating optimal conditions for MAA production [21], while 3 mM corresponded to the maximum nitrate concentration naturally available in the fishpond effluents’. We agree that testing additional nitrogen concentrations and alternative nitrogen sources would provide valuable insights. However, our objective is to advance an IMTA framework, integrating aquatic organisms to recirculate water and valorize fish effluents as nutrient inputs for P. dioica, focusing on concentrations naturally present in the system. At present, Chelon labrosus is the only fish species available in our facilities, but future studies could include other aquatic organisms once additional cultures become accessible at our research center.

Other remarks:

Tables in figure 2 too small

Response: We agree, and have changed it.

number of replicates is not offen given

Response: We agree, and have added this information throughout the text.

Figure 3 : no SD given for PE:PC

Response: We appreciate the reviewer’s comment. However, since PE:PC represents a ratio between two phycoerythrin and phycocyanin rather than an independent measurement, standard deviation cannot be directly calculated for the ratio itself.

Table 5: precise if it is % dry or fresh weight.

Response: We agree, and have added this information. The correct is: % dry weight.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a valuable investigation into the morphological, physiological, and biochemical changes of the red macroalgae Porphyra dioica depending on nitrate source and concentration under UVR. This research may potentially improve the future avenues in IMTA cultivation and thus promote environmental sustainability.

The study is solid and comprehensive, no additional experiments are required. However, there is a need for improvement in presenation clarity.

Introduction: The current state of IMTA research needs to be better articulated. Furthermore, "potential health benefits" in Line 41 are too vague. The authors should specify which benefits they are talking about with supporting references.

Methods: The Methods section of the manuscript has been flagged by the automatic journal system for high percentage of similiarity with authors' previous studies. This section must be thoroughly rephrased and rewritten, even when describing standard protocols.

To ensure the reproducibility of the research, the callibration curves for NO₃^-, gallic acid, and Trolox should be included in the Supplementary Materials.

Moreover, the formulas throughout the manuscript (e.g., Lines 135, 151, 152, 160, 167, 168, 222) do not conform to the journal's style and must be revised by authors.

Results: Figure 1 and Lines 270-271, 465 - the depigmentation/discoloration mentioned in main body of the manuscript is not visible in the provided microphotographs. The authors should consider to replace these images with clearer examples that will demonstrate changes in coloration of thalli.

The very large standard deviation (SD) bars in Figure 2 may undermine the reliability of the conclusions. The authors should provide written reasons in the main body of the manuscript regarding SD or re-run the experiment, if possible.

Discussions: The discussion frequently diverts to comparisions with strawberries (e.g., Lines 522-523, 545), which is not directly relevant to the research. The focus should be on comparing the observed effects with those reported for other macroalgae used in IMTA or other sustainable cultivation systems. In my opinion, to significantly improve the manuscript authors should discuss their fundings with data on other macroalgae, and, if necessary, create a comparative table that will place their results side-by-side with previously published data.

First lines (427-431) read like a conclusions and should be integrated into the actual conclusions section.

The authors discuss chlorophyll and carotenoids but did not measure them directly. There is a mention of "yellowish coloration observed in thalli" in Lines 495-498, but it was not obvious from Figure 1. A justification of this choice should be provided.

Conclusions: Currently this section is too brief. Conclusions should be expanded to provide more detailed and specific summary of key fundings and their implications for future IMTA development.

Specific comments: Some revision of grammatical errors, typos, and formating is needed.

For example:

Line 4 - the species name Porphyra dioica should be in cursive in the title.

Line 18, 75 - spaces or punctuation marks in the light intensity units (e.g., photons m^-2 s^-1)

Line 45 - correct "diabetic" to "diabetes"

Line 68 - correct the full name of Institute for clarity

Line 104 - standardize the terminology for replicates throughout the manuscript (triplicates, or n=3)

Line 183 - "SC" was not used in the main body of the manuscript

Line 235 - the reference format here is incorrect and must be changes to match the journal's style

Line 272 - "was greatest" should be reworded

Line 299 - the phrasing "the lowest in this with 3 mM" is unclear and should be reworded

Line 422-433 - check the brackets

Line 479 - "to affect negatively electron transport" should be reworded

Line 480 - "has not negative effect" should be reworded

Lines 704-705 (Ref 54) - the reference format is incorrect and must be changes to match the journal's style

Figures and Tables captions ("different nitrate sources and concentrations and UVR") may be reworded as in Lines 292-293 ("different nitrate sources and concentrations with UVR")

The manuscript could become a strong contribution to the field, but without the revision the clarity and presenation will be compromised.

Author Response

The study is solid and comprehensive, no additional experiments are required. However, there is a need for improvement in presentation clarity.

Response: We agree, and have improved this section.

Methods: The Methods section of the manuscript has been flagged by the automatic journal system for high percentage of similarity with authors' previous studies. This section must be thoroughly rephrased and rewritten, even when describing standard protocols.

Response: We agree, and have revised and rewritten the methodology in full.

To ensure the reproducibility of the research, the calibration curves for NO₃^-, gallic acid, and Trolox should be included in the Supplementary Materials.

Response: We agree, and have added the graphics in the Supplementary Materials.

Moreover, the formulas throughout the manuscript (e.g., Lines 135, 151, 152, 160, 167, 168, 222) do not conform to the journal's style and must be revised by authors.

Response: We agree, and have corrected all formulas.

Response: We appreciate your observation, and to improve understanding of the color changes, we added information in the Results section describing the color characteristics analyzed using Photoshop software.

Discussions: The discussion frequently diverts to comparisons with strawberries (e.g., Lines 522-523, 545), which is not directly relevant to the research. The focus should be on comparing the observed effects with those reported for other macroalgae used in IMTA or other sustainable cultivation systems. In my opinion, to significantly improve the manuscript authors should discuss their fundings with data on other macroalgae, and, if necessary, create a comparative table that will place their results side-by-side with previously published data.

Response: We agree and have revised the discussion to include comparisons with other macroalgae used in IMTA and sustainable cultivation systems.

First lines (427-431) read like a conclusion and should be integrated into the actual conclusions section.

Response: We agree, and have changed it.

Response: Thank you for your observation. We did not perform chlorophyll or carotenoid quantification because the main objective of our research group is to obtain aqueous extracts suitable for nutricosmetic applications. Therefore, we exclusively use non-toxic solvents such as water in all extraction procedures, which do not allow the extraction and quantification of chlorophylls and carotenoids. In the results section, we have added information on the color characteristics based on the analysis performed using Photoshop software.

Conclusions: Currently this section is too brief. Conclusions should be expanded to provide more detailed and specific summary of key fundings and their implications for future IMTA development.

Response: We agree, and have improved it.

Specific comments: Some revision of grammatical errors, typos, and formatting is needed.

For example:

Line 4 - the species name Porphyra dioica should be in cursive in the title.