An Overview of Bioproducts from Wastewater-Grown Microalgae: Recent Advancements, Economic and Feasibility Concerns

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

This review addresses a relevant topic, compiles a broad range of recent literature but with important gaps. However, the manuscript is largely descriptive and would benefit from a more critical and mechanistic discussion throughout. The inclusion of schematic figures and summary tables is strongly recommended to improve readability. In addition, several sections should be expanded to better explain the biological mechanisms underlying the reported results, critically compare the cited studies, discuss the limitations of current technologies, and provide clearer perspectives for future research and industrial implementation.

Majors:

-The manuscript would greatly benefit from the inclusion of one or two schematic figures. At present, the review is entirely text-based, making it difficult for readers to synthesize the large amount of information presented. I strongly recommend incorporating, an overview of the complete microalgae-based wastewater biorefinery, from wastewater sources to biomass valorization; a schematic illustration of the main mechanisms involved in microalgae-mediated wastewater remediation, including nutrient assimilation, carbon fixation, heavy metal removal, and microalgae–bacteria interactions, or a flowchart summarizing the major techno-economic and life cycle assessment bottlenecks across the entire process.

-Also, the review would benefit from the inclusion of tables to synthesize the extensive information presented throughout the review. I recommend for instance adding one table comparing the different wastewater types, microalgal species, and treatment efficiencies.

-L49: “heavy metals” molybdenum is one of the most important and C. reinhardtii has emerged as the leading microalgal model for studying molybdenum homeostasis through the identification of two novel ABC-type transporters that may be involved in his detoxification. Please, consider discussing this aspect.

-Throughout the manuscript, little consideration is given to Chlamydomonas, the most widely used model microalga in research, where it has been shown to be essential for bioremediation and bioproduct production. The few times it is mentioned, it is done incorrectly, as in line 543: "Chllorella reinhardtii"? Please correct this.

-L97…: Several microalgal species are cited throughout this section; however, the manuscript does not explain why these organisms are particularly suitable for wastewater treatment. A brief discussion of the physiological characteristics that make species highly effective (rapid growth, tolerance to wastewater conditions…) is necessary, please.

-L140…: It is  reported nutrient removal efficiencies without discussing the biological mechanisms responsible. Please explain why different microalgal species exhibit distinct removal capacities, considering factors such as metabolic flexibility, mixotrophic growth, nutrient uptake ….

-L202…: This is just an example but is over all the manuscript, it is  mainly lists nutrient removal efficiencies without explaining the factors responsible for the observed differences. A brief discussion of the main parameters affecting treatment performance would considerably add critical value, please.

-L253….:It is introduced  the mechanisms of nutrient assimilation, but lacking  mechanistic explanation of the different pathways involved, please improve.

-L304: The section on "Microalgal-bacterial consortia" is critical and, in my opinion, should be discussed in greater depth given its importance. For example, microalgal and nitrogen-fixing bacterial consortia have significant biotechnological potential and deserve further consideration. This consortia will improve remove contamination in low nitrogenated wastewater.

-L312: "Nitrogen and phosphorus", it should also be mentioned that microalgae are also able to assimilate organic nitrogen sources, such as amino acids, through extracellular L-amino acid oxidase 1. Please consider

-L402…: Here, several studies on hydrothermal liquefaction are summarized, but  lacks a critical comparison of the main limitations preventing industrial implementation, please improve.

-L429….:It is  reported biodiesel production but without a critical discussion of the major limiting factor for their commercial feasibility.

-L539…: biobutanol is a promising alternative to bioethanol but does not critically discuss its limitations.

-L690…: Identifies biomass composition as a major factor affecting processing but does not critically analyze how wastewater variability influences quality.

- A potential drawback of using microalgae is that they can produce greenhouse gases, such as nitrous oxide, highly dependent on  nitrate containing media. Please consider

Author Response

We have responded to the reviewer 1's feedback in the attached word document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “An overview of bioproducts from wastewater grown microalgae: recent advancements, economic and feasibility concerns” was reviewed. This is an informative and well organized review. Here are some suggestions/revisions:

 

This review lacks the involvement of representative figures and tables that facilitate the ease delivery of concepts and contents to the readers.

- 2.1 Wastewater streams: A valuable comparison could be introduced between different mentioned Wastewater streams.

- In addition, a table should be constructed illustrating examples of different algal species enriching these different streams.

- 2.2 Nutrient Assimilation, Biosorption, and Microbial Symbiosis: A representative figure could explain each nutrient assimilation properly.

- Heavy metals remediation was not introduced  conveniently,  a broad section should be involved.

- 4. Techno-Economic Analysis and Life Cycle Assessment of Bioproducts Obtained from Microalgae-Based Wastewater Treatment: Should be supported with representative diagram presenting Techno-economic analysis (TEA) and life cycle assessment (LCA) tools.

Author Response

The response to Reviewer 2's feedback can be found in the attached word document.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my comments satisfactorily. However, several points still require revision.

-L48: Current text already discusses N2O, nitrite and NO, but nitrate-containing media are not explicitly emphasized enough. Consider that, N2O emissions should be considered as a potential environmental drawback when evaluating nitrate-rich wastewater streams, please, cite the proper reference.

-L483: Incomplete sentence: “In the final step, glutamine synthetase (GS) drives the ATP [77]” replace with something like that: Consequently, all inorganic nitrogen forms must be reduced to intracellular ammonium (NH4+) before metabolic assimilation. In the final step, ammonium is incorporated into amino acids mainly through the GS/GOGAT pathway. Glutamine synthetase (GS) catalyzes the ATP-dependent incorporation of ammonium into glutamate to form glutamine, while glutamate synthase (GOGAT) transfers the amide group of glutamine to 2-oxoglutarate, producing two molecules of glutamate. Please

L551: “Investigations utilizing the green model microalga Chlamydomonas reinhardtii have successfully elucidated key molecular components governing molybdenum (Mo) homeostasis, encompassing both molybdate transport mechanisms and molybdoenzyme activities.” The authors have correctly revised this section, but they have not added any references. These should be Tejada-Jiménez 2023 and Leon-Miranda 2025. Please

-L563: The authors added a general section on algae-bacteria symbiosis, but it does not specifically discuss nitrogen-fixing bacterial partners or their relevance for low-nitrogen wastewater. Please add something like this: consortia between microalgae and nitrogen-fixing bacteria deserve particular attention for wastewater streams with low nitrogen availability. Diazotrophic bacteria can provide biologically fixed nitrogen to the algal partner, while microalgae supply oxygen, organic carbon, and photosynthetically derived metabolites that support bacterial activity. These mutualistic associations may improve biomass productivity, reduce the need for external nitrogen supplementation, and enhance the overall sustainability of wastewater-based microalgal biorefineries, and the proper citation. And the proper citation please.

-2.2.3.4 Algae-bacteria symbiosis must be: 2.2.3.5 Algae-bacteria symbiosis

-L875….: limitation of biobutanol is still too superficial. Consider something like that: In the specific case of biobutanol, additional limitations include low solvent yields, product toxicity to fermenting microorganisms, and the variability of wastewater-grown biomass composition, all of which hinder process standardization and commercial feasibility

Author Response

Thank you again for the constructive feedback, the issues which were raised by the reviewer were modified, and the response to each observation can be found in the attached word document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for proper response.

Author Response

The authors would like to thank again the reviewers for their constructive feedback which improved the quality of our work.