Bioprospecting Native Oleaginous Microalgae for Wastewater Nutrient Remediation and Lipid Production: An Environmentally Sustainable Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I read with pleasure your manuscript with a challenging, significant and actual subject in the field of wastewater treatment systems with secondary (biological) and tertiary (mechanical-chemical, e.g. reduction study of total oxidated P-based and oxidated / reduced N-based species content for nutrient remediation) steps in association with the selection of best biological combinations (oleaginous microalgal consortia) for highest yield of bio-energetical products' production (lipid acumulation and separation for bio-oil / biofuel production).

I like the manuscript which fulfills all scientific requirements for publication, being actual, clear, having validated data, new scientific findings by advanced analysis technics and testing control experiments in real operating state which permited high efficiency of pollutants' retaining, with final nutrient reduction/remediation and lipid accumulation into the produced biomass further valorized.

For publication of your manuscript, there are required a few minor corrections such as

(i) page 3, line 113 - delete of the first 'and' and replace with ';'

(ii) page 3 - if is possible to insert details (in bracket) with the main unitary operations and processes and corresponding treatment equipments/machinery involved in the preliminary treatment, primary and secondary clarification and even biological treatment regime.

(iii) Please insert a final section with Conclusions which actually is missing.

(iv) At the final section of References, a few corrections are required including a uniform writing style of journal names, e.g.,

(a) italic style of journal name for refs. 4,9,12,15,18,20,22-24, 28.29,32,34,36,37,38,40, 42,43,47,49,51,56,59,60) and

(b) select abbreviated or complete forms of the same journal name not both, as for Scientific Reports or Sci. Rep.

As concluding remark, your manuscript is acceptable for publication due to the numerous and various experimental data findings, selection of the best combinations of microalgal consortia for wastewater treatment and energetical valorization of developed/produced biomass, final total N and P species (nutrient remediation) reduction until legal admissible limits and lipid accumulation for bio-energy product valorization (as potential biofuel).

Manuscript Reviewer

Author Response

Reviewer #1:

I read with pleasure your manuscript with a challenging, significant and actual subject in the field of wastewater treatment systems with secondary (biological) and tertiary (mechanical-chemical, e.g. reduction study of total oxidated P-based and oxidated / reduced N-based species content for nutrient remediation) steps in association with the selection of best biological combinations (oleaginous microalgal consortia) for highest yield of bio-energetical products' production (lipid acumulation and separation for bio-oil / biofuel production).

I like the manuscript which fulfills all scientific requirements for publication, being actual, clear, having validated data, new scientific findings by advanced analysis technics and testing control experiments in real operating state which permited high efficiency of pollutants' retaining, with final nutrient reduction/remediation and lipid accumulation into the produced biomass further valorized.

Author’s Response: We greatly appreciate your time in reviewing our manuscript which has facilitated improvement of our study. Per your instructions, we provide a point-by-point response to your comments/ suggestions and hope that this version is acceptable for publication.

For publication of your manuscript, there are required a few minor corrections such as

(i) page 3, line 113 - delete of the first 'and' and replace with ';'

Author’s Response: The first ‘and’ has been deleted and a semi-colon has been inserted to connect the objectives.

(ii) page 3 - if is possible to insert details (in bracket) with the main unitary operations and processes and corresponding treatment equipments/machinery involved in the preliminary treatment, primary and secondary clarification and even biological treatment regime.

Author’s Response:  Additional context to the facility from where samples were collected for this study have been provided on page 3, lines 87-101.

(iii) Please insert a final section with Conclusions which actually is missing.

Author’s Response:  A conclusion section at the end of the manuscript has been added.

(iv) At the final section of References, a few corrections are required including a uniform writing style of journal names, e.g.,

(a) italic style of journal name for refs. 4,9,12,15,18,20,22-24, 28.29,32,34,36,37,38,40, 42,43,47,49,51,56,59,60) and

(b) select abbreviated or complete forms of the same journal name not both, as for Scientific Reports or Sci. Rep.

Author’s Response: All references have been formatted in accordance with the journal style.

As concluding remark, your manuscript is acceptable for publication due to the numerous and various experimental data findings, selection of the best combinations of microalgal consortia for wastewater treatment and energetical valorization of developed/produced biomass, final total N and P species (nutrient remediation) reduction until legal admissible limits and lipid accumulation for bio-energy product valorization (as potential biofuel).

Author’s Response: We greatly appreciate your time in reviewing our manuscript which has facilitated improvement of our study. Hope that the revised version is acceptable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled "Bioprospecting Native Oleaginous Microalgae for Wastewater Nutrient Remediation and Lipid Production: An Environmentally Sustainable Approach" is an interesting work on the bioprospected oleaginous microalgal consortia from a wastewater holding tank, aiming to identify strains capable of both nutrient remediation and lipid accumulation.
The work is well structured.
Some comments are:
1) Since the study is based on samples from a single facility (T.P. Smith WRF), could the authors comment on how this limited spatial and temporal scope might affect the broader applicability or generalization of their findings?
2) The use of Fluorescence Activated Cell Sorting (FACS) is intriguing. Could the authors elaborate on whether this method was validated for these native mixed consortia—for example, was the fluorescence intensity confirmed to correlate quantitatively with lipid content across the species present?
3) Regarding the nutrient removal experiments, were environmental parameters such as light intensity, pH, temperature, and inoculum size carefully controlled and kept consistent among all consortia? This information would be useful to ensure the observed differences are biologically meaningful.
4) The Sulfo-Phospho-Vanillin (SPV) method is a common approach for lipid quantification, but it can be influenced by pigments and carbohydrates. Could the authors justify its suitability for complex mixed consortia and discuss any potential limitations or corrections applied?
5) The reported differences in performance among consortia (C3, C4, C9) are interesting. Were these differences statistically tested, or are they mainly based on visual interpretation of the data? Including statistical analysis would add robustness to these comparisons.
6) In Figures 7–9, the observed nutrient removal trends are attributed to biological activity. However, could abiotic mechanisms, such as adsorption onto biomass or chemical precipitation, also play a role? A brief discussion on this possibility would clarify the interpretation.
7) Considering the potential application of these consortia in wastewater treatment systems, have the authors evaluated any biosafety or ecological risks associated with the introduction or re-release of concentrated microbial consortia into natural or engineered environments?

Author Response

Reviewer #2:

The manuscript entitled "Bioprospecting Native Oleaginous Microalgae for Wastewater Nutrient Remediation and Lipid Production: An Environmentally Sustainable Approach" is an interesting work on the bioprospected oleaginous microalgal consortia from a wastewater holding tank, aiming to identify strains capable of both nutrient remediation and lipid accumulation.
The work is well structured.

Author’s Response: 

Some comments are:
1) Since the study is based on samples from a single facility (T.P. Smith WRF), could the authors comment on how this limited spatial and temporal scope might affect the broader applicability or generalization of their findings?
Author’s Response: This is a relevant point and we have added a new “conclusion” section that includes this cautionary note on page 17, lines 484-497.

2) The use of Fluorescence Activated Cell Sorting (FACS) is intriguing. Could the authors elaborate on whether this method was validated for these native mixed consortia—for example, was the fluorescence intensity confirmed to correlate quantitatively with lipid content across the species present?
Author’s Response:  We have provided a note of caution to address this valid point on page 6, lines 271-283. We stated that “The application of Fluorescence Activated Cell Sorting (FACS) in this study was primarily aimed at high-throughput isolation of lipid-rich microalgal cells from complex wastewater consortia. While BODIPY 505/515 staining is widely recognized as a reliable proxy for intracellular lipid content in microalgae, we acknowledge that fluorescence intensity can vary among species due to differences in cell wall permeability, dye uptake, and lipid composition. In this work, the correlation between fluorescence intensity and lipid content was indirectly validated by subsequent Sulfo-Phospho-Vanillin (SPV) assays on sorted isolates, which confirmed that consortia exhibiting higher fluorescence generally had greater lipid yields. However, a quantitative calibration across all species present was not performed, and this represents a limitation of the current approach. Future studies should incorporate species-specific calibration curves or complementary imaging techniques to strengthen the quantitative relationship between fluorescence signal and lipid content in mixed consortia”.

3) Regarding the nutrient removal experiments, were environmental parameters such as light intensity, pH, temperature, and inoculum size carefully controlled and kept consistent among all consortia? This information would be useful to ensure the observed differences are biologically meaningful.
Author’s Response: Yes, environmental parameters were carefully controlled to ensure comparability among consortia, as stated on page 16, lines 475-488. We agree that even though these parameters were standardized, micro-scale variations in consortial composition could still influence performance. Future work could incorporate real-time monitoring of dissolved oxygen and CO₂ to further refine environmental control and better understand metabolic dynamics.

4) The Sulfo-Phospho-Vanillin (SPV) method is a common approach for lipid quantification, but it can be influenced by pigments and carbohydrates. Could the authors justify its suitability for complex mixed consortia and discuss any potential limitations or corrections applied?
Author’s Response: This has been clarified on page 10, lines 312-322. We state that “Note that the Sulfo-Phospho-Vanillin (SPV) assay was selected for lipid quantification due to its simplicity and compatibility with high-throughput screening. However, we acknowledge that SPV can be influenced by interfering compounds such as pigments and carbohydrates, which are abundant in mixed microalgal consortia. To mitigate these effects, samples were thoroughly washed and dried prior to analysis, and lipid estimates were normalized against a triolein calibration curve to improve accuracy. Despite these precautions, SPV remains a colorimetric method that provides semi-quantitative results rather than absolute lipid content, particularly in heterogeneous samples. Future work should incorporate complementary techniques such as gravimetric extraction or gas chromatography–mass spectrometry (GC-MS) to validate SPV results and ensure robust quantification across diverse species”.

5) The reported differences in performance among consortia (C3, C4, C9) are interesting. Were these differences statistically tested, or are they mainly based on visual interpretation of the data? Including statistical analysis would add robustness to these comparisons.
Author’s Response: The differences in nutrient removal performance among consortia C3, C4, and C9 were visually evident in the depletion curves; however, these were also subjected to statistical analysis to confirm significance. The statistical analysis has now been added on page 17, lines 524-544 and a new Figure 9 has been added to reflect the statistical analysis.

6) In Figures 7–9, the observed nutrient removal trends are attributed to biological activity. However, could abiotic mechanisms, such as adsorption onto biomass or chemical precipitation, also play a role? A brief discussion on this possibility would clarify the interpretation.
Author’s Response: We agree, and this has been added on page 17, lines 545- 555. We state that “One final cautionary interpretation on the nutrient removal trends observed in Figures 7–9; we believe that these are primarily attributed to biological activity within the algal–microbial consortia, however, abiotic mechanisms may also contribute. Adsorption of phosphate and ammonium ions onto cell surfaces or extracellular polymeric substances, as well as chemical precipitation of phosphorus under certain pH and ionic conditions, are known to occur in wastewater environments. While our experimental setup maintained relatively stable pH and did not include chemical additives, these processes cannot be completely excluded. Future studies should incorporate abiotic controls (e.g., sterilized biomass or filtered wastewater) to quantify the relative contributions of biological versus physicochemical mechanisms, thereby improving interpretation of nutrient removal pathways”.

7) Considering the potential application of these consortia in wastewater treatment systems, have the authors evaluated any biosafety or ecological risks associated with the introduction or re-release of concentrated microbial consortia into natural or engineered environments?

Author’s Response: We agree, and this has been added on page 17, lines 557- 567. We state that “It should also be noted that the potential application of these consortia in wastewater treatment systems raises important biosafety and ecological considerations. Although the strains were isolated from municipal wastewater and are therefore native to such environments, concentrating and reintroducing them could alter microbial community dynamics or facilitate unintended dispersal of opportunistic species. Risks include horizontal gene transfer, proliferation of antibiotic resistance genes, and ecological imbalance if consortia escape into natural water bodies. While our study did not specifically evaluate these risks, future work should incorporate comprehensive biosafety assessments, including pathogenicity screening, antibiotic resistance profiling, and ecological impact modeling. Regulatory compliance with local and federal guidelines (e.g., EPA and state environmental agencies) will be essential before any large-scale deployment”.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is interesting, as it discusses the indigenous oil-rich microalgae for wastewater treatment and oil generation. The method of screening is also quite new considering most of the time isolation and manual screening are preferred. However, several comments for the manuscript shall be considered for the improvement of quality and clarity.

1. Authors shall consider to put the wastewater characteristics as one of the main considerations. It is also important to show it in the result why the authors use ammonia/nitrate as parameter.
2. It is interesting the both eukaryotic and prokaryotic are blooming at the same time. what is the pH and how does the pH role this community?
3. The figure presented in low quality and monotonous.
4. The method to do the lines for nutrient removal needs to be explained. Does it include removal rate model or not? On day 10 phosphate level is zero?
5. Interesting to see that the ammonia was presented and not ammonium, indicating the relatively alkaline pH. and this often become toxic to microalgae. How the authors address this phenomenon.
6. It is difficult to draw any conclusion from the manuscript.
7. there is no significant discussion regarding results that actually address what authors explained in the introduction about oil production.

Overall the manuscript need improvement and correction to make it suitable for publication. 

Author Response

Reviewer #3:

The manuscript is interesting, as it discusses the indigenous oil-rich microalgae for wastewater treatment and oil generation. The method of screening is also quite new considering most of the time isolation and manual screening are preferred. However, several comments for the manuscript shall be considered for the improvement of quality and clarity.

Author’s Response:  We greatly appreciate your time in reviewing our manuscript which has facilitated improvement of our study.

Authors shall consider to put the wastewater characteristics as one of the main considerations. It is also important to show it in the result why the authors use ammonia/nitrate as parameter.

Author’s Response: Typical influent wastewater characteristics at the Thomas P. Smith Water Reclamation Facility (TPSWRF), Tallahassee, Florida is now shown in table SI-1 and page 4 lines 148-153. Values represent approximate ranges for municipal wastewater based on facility reports and literature, thus providing context for nutrient removal experiments and justify the selection of ammonia and nitrate as key indicators, as they are dominant nitrogen species in municipal wastewater and critical for assessing treatment performance.

It is interesting the both eukaryotic and prokaryotic are blooming at the same time. what is the pH and how does the pH role this community?

Author’s Response: The pH of the wastewater during enrichment and nutrient removal experiments was maintained near neutral (6.8–7.2), which is typical for municipal wastewater and conducive to both algal and bacterial growth. Neutral pH supports photosynthetic activity in microalgae while also favoring bacterial metabolic processes such as nitrification and organic matter degradation. At this range, carbon dioxide availability and bicarbonate buffering are optimal for algal photosynthesis, while ammonia remains in a form that bacteria can assimilate or oxidize. Extreme pH shifts could disrupt this balance- acidic conditions inhibit nitrifiers, while alkaline conditions can reduce CO₂ availability for algae and promote chemical precipitation of phosphorus. Thus, maintaining stable pH likely facilitated the observed co-bloom and synergistic interactions between eukaryotic and prokaryotic members of the consortia.

The figure presented in low quality and monotonous.

Author’s Response: We have revised the figures, but the system reduces the quality during the conversion process. We can work with the publisher after the manuscript is accepted to further improve the figure quality.

The method to do the lines for nutrient removal needs to be explained. Does it include removal rate model or not? On day 10 phosphate level is zero?

Author’s Response: The nutrient removal trends shown in Figures 7–9 were generated by plotting measured concentrations of phosphate, nitrate, and ammonia over a 12-day incubation period. Samples were collected daily, and concentrations were determined using ICP-OES for phosphate, UV spectrophotometry for nitrate, and a handheld photometer for ammonia. The plotted lines represent observed values connected sequentially; no kinetic removal rate model (e.g., first-order or Monod) was applied in this study. Therefore, the curves are descriptive rather than predictive.

Regarding the observation that phosphate levels approach zero by day 10, this reflects the experimental measurements for consortium C9, which exhibited near-complete phosphorus depletion under the controlled conditions. However, residual phosphorus may still exist below detection limits. Future work could incorporate kinetic modeling to estimate removal rates and better understand underlying mechanisms.

Interesting to see that the ammonia was presented and not ammonium, indicating the relatively alkaline pH. and this often become toxic to microalgae. How the authors address this phenomenon.

Author’s Response: The presentation of ammonia rather than ammonium in our results reflects the relatively alkaline pH of the wastewater (6.8–7.5), which favors the un-ionized form (NH₃). This is noteworthy because free ammonia is known to be toxic to many microalgal species, potentially inhibiting photosynthesis and growth at elevated concentrations. In our experiments, toxicity was mitigated by two factors: (i) the presence of mixed consortia, which likely facilitated ammonia assimilation through synergistic interactions with bacteria, and (ii) controlled aeration and light conditions that promoted rapid uptake and conversion of ammonia into biomass. No visible growth inhibition was observed during the 12-day incubation, suggesting that the consortia were either tolerant to the tested concentrations or adapted to wastewater conditions. Future studies should quantify free ammonia toxicity thresholds for these consortia and explore strategies such as pH adjustment or staged nitrogen removal to minimize inhibitory effects in large-scale applications. This is stated on page 16, lines 504-516.

It is difficult to draw any conclusion from the manuscript.

Author’s Response: A new conclusion section has been added to address your point.

there is no significant discussion regarding results that actually address what authors explained in the introduction about oil production.

Author’s Response: You’re absolutely correct—there’s a gap between what was promised in the Introduction (oil/lipid production potential) and what is discussed in the Results section. We have addressed this on page 11, lines 330-337. Note the lipid accumulation results confirm that the isolated consortia can serve as dual-purpose agents- removing nutrients while generating biomass enriched in lipids.

Overall the manuscript need improvement and correction to make it suitable for publication.

Author’s Response: We have significantly revised the manuscript and hope it is now suitable for publication.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have improved the manuscript accordingly.

 

Author Response

As stated by the reviewer, "The authors have improved the manuscript accordingly". We believe there were no further suggested revisions by reviewer #2 and that the manuscript is ready for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made several improvement. However, there are several comments needs to be addressed further.

1. Ln 73, not appropriate citation.
2. A photo or map shall help to give the context of the introduction of the wastewater plant not in the appendices and please provide a proper quality of any figure or aerial photo.
3. Figure 1 shall be ordered by the consortium name.
4. Quality of the figures not improved. There is also inconsistency in between figures (See fig 6B and other figures)
5. The authors shall consider to restructure the figure or put it in more simple display to ensure clarity  of the data presentation.
6. Fig 9 caption shall not contain any method explanation.
7. Where are the models come from for the nutrient reduction? Does the measurement or experiment have replication? where is the STDEV or SE or SEM in figure 7-9? These figures can be combined in one figure with three panels.
8. Ln 441. I do not think it is necessary to refer which statistic authors used to reach the conclusion or to compare the data, explain straight to the result whether it is significantly different or not.
9. Conclusion needs to be reconstructed.

Author Response

Please review the revised manuscript uploaded and we hope that it covers all your comments/ suggestions. 

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have been revised the manuscript accordingly, the content has been improved. Figure 3 to six seems to be monotonous still and it would be great if the authors can merge these figures as well to more simple yet understandable graphs.

Author Response

Figure 3 and associated figures report on the taxonomic and gene functions identified within the different consortia from our study. We believe that this is the best representation of our data and hope this is agreeable to the reviewer. Thanks!