Bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs) in Aqueous Environments: A Review of Biofiltration, Biosorption, and Biodegradation Strategies Using Living Fungal Mycelium

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses fungal-based strategies for polycyclic aromatic hydrocarbon (PAH) remediation, which is timely and relevant. The structure is clear, but several issues require substantial revision before the work can be accepted. These include inconsistencies in terminology and table numbering, insufficient mechanistic detail, lack of quantitative thresholds, weak integration of recent findings, and absence of practical design criteria. The manuscript would also benefit from strategic citations to important recent literature to reinforce its claims.

Title and Abstract

    1.The abstract ends vaguely with “lays the foundation for future material research.” Please specify the unique contribution of this review (e.g., integrating fungal biofilms and Engineered Living Materials for PAH remediation).
    2.In the abstract and introduction, clarify that fungal-based systems are not just complementary to physical/chemical methods, but can provide multifunctional remediation with lower resource input.

Introduction

   3. Add a concise statement on the innovation and boundary of this review (e.g., focusing on fungal matrices as living filtration membranes) and condense the excessive repetition on the hazards of PAHs.

Section 3 – Biofiltration

    4. The section currently mentions “material properties influence attachment,” but does not provide quantitative criteria. Please cite Lu et al., 2025 （https://doi.org/10.1016/j.cej.2025.16165）here, which systematically demonstrated that liquid-holding capacity and BET surface area of lignocellulosic carriers directly determine biofilm productivity. This would strengthen the discussion by linking fungal carrier selection to measurable physical parameters.

Section 5 – Biosorption

   5. Statements on functional groups and π–π interactions should be expanded by discussing competitive adsorption, sorption isotherms, and kinetic models. Please add specific ranges of Freundlich/Langmuir constants or pseudo-second-order rate constants from recent literature.

Section 12 – Discussion

   7. Revise “fungal membranes largely uninvestigated” to “under-explored” and include a comparative table summarizing existing fungal biofilm or mycelial membrane studies, highlighting gaps specific to PAHs.

Tables and Figures

   9. Table numbering is inconsistent (e.g., Table 6 is referenced before appearing, Table 4 has a placeholder title). Please renumber and provide full captions, including methods, units, and references. Figures should have consistent design, and Figure 4 should specify typical pore sizes or cutoff values for membrane stages.

Others

   10. In the introduction or methods description, add a PRISMA-style flow diagram of the literature search and inclusion/exclusion criteria. This will improve reproducibility of your review process.

   11. Add a design checklist at the end of Section 12 summarizing: (i) influent water quality windows (e.g., DOM thresholds), (ii) carrier selection criteria (e.g., BET and liquid-holding capacity), (iii) microbial partnerships (e.g., enhancement rate concept), and (iv) key operational indicators (e.g., flux decline, ROS levels).
 

 

Author Response

We sincerely thank the reviewer for their thoughtful and constructive feedback, which has significantly improved the clarity and contribution of our manuscript. Below, we provide a point-by-point response. All revisions in the manuscript are highlighted.

 

Comment 1. The abstract ends vaguely with “lays the foundation for future material research.” Please specify the unique contribution of this review (e.g., integrating fungal biofilms and Engineered Living Materials for PAH remediation).
Response 1. We appreciate this suggestion. We have revised lines 9–15 of the abstract to specify the unique contribution of this review, particularly in relation to ELM applications.

Comment 2. In the abstract and introduction, clarify that fungal-based systems are not just complementary to physical/chemical methods, but can provide multifunctional remediation with lower resource input.
Response 2. We agree. The text has been revised in the abstract (lines 9–15) and in the introduction (lines 53–55) to emphasize the multifunctional role of fungal systems in remediation.

Comment 3. Add a concise statement on the innovation and boundary of this review (e.g., focusing on fungal matrices as living filtration membranes) and condense the excessive repetition on the hazards of PAHs.
Response 3. We agree. We have revised the abstract (lines 9–15) and introduction (lines 55–58) to clearly state the scope and innovation of this review, while also reducing redundant discussion of PAH hazards.

Comment 4. The section currently mentions “material properties influence attachment,” but does not provide quantitative criteria. Please cite Lu et al., 2025 （https://doi.org/10.1016/j.cej.2025.16165）here, which systematically demonstrated that liquid-holding capacity and BET surface area of lignocellulosic carriers directly determine biofilm productivity. This would strengthen the discussion by linking fungal carrier selection to measurable physical parameters. 
Response 4. Thank you for this suggestion. Unfortunately, we were unable to access the cited article, as the DOI currently leads to an error and alternative database searches were inconclusive. The reference appears highly relevant, and we would be grateful if the title could be shared to allow us to include it.

Comment 5. Statements on functional groups and π–π interactions should be expanded by discussing competitive adsorption, sorption isotherms, and kinetic models. Please add specific ranges of Freundlich/Langmuir constants or pseudo-second-order rate constants from recent literature.
Response 5. We found this comment highly relevant. Section on π–π interactions has been expanded (lines 316–337) to include competitive adsorption, sorption isotherms, and kinetic models, along with representative parameter ranges from recent studies.

Comment 6. Revise “fungal membranes largely uninvestigated” to “under-explored” and include a comparative table summarizing existing fungal biofilm or mycelial membrane studies, highlighting gaps specific to PAHs.
Response 6. The term has been revised to “under-explored” (line 182). Additionally, a comparative table has been added to summarize fungal biofilm and mycelial membrane studies and highlight research gaps relevant to PAH remediation.

Comment 7. Table numbering is inconsistent (e.g., Table 6 is referenced before appearing, Table 4 has a placeholder title). Please renumber and provide full captions, including methods, units, and references. Figures should have consistent design, and Figure 4 should specify typical pore sizes or cutoff values for membrane stages.
Response 7. Thank you for noting these inconsistencies. We have renumbered all tables and figures, corrected captions (e.g., Table 5, p. 17), and ensured consistency in design. Figure 5 (previously Figure 4) has been updated to specify pore size ranges for different membrane types.

Comment 8. In the introduction or methods description, add a PRISMA-style flow diagram of the literature search and inclusion/exclusion criteria.
Response 8. We agree. A PRISMA-style diagram has been added on page 4 of the introduction to clarify the literature selection process.

Comment 9. Add a design checklist at the end of Section 12 summarizing: (i) influent water quality windows (e.g., DOM thresholds), (ii) carrier selection criteria (e.g., BET and liquid-holding capacity), (iii) microbial partnerships (e.g., enhancement rate concept), and (iv) key operational indicators (e.g., flux decline, ROS levels).
Response 9. We agree. Section 12 has been expanded (line 503- 523) with a design checklist that incorporates influent water quality and the State of the art of concentration and types of PAHs effectively degraded. We have stated the constraints in a multi-solute scenario, optimal pH and temperature conditions, the most common fugal carriers, the importance of monitoring the water flux through the membrane and finally the potential of microbial partnership in remediation.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments:

- Can you describe more specifically the sources of PAHs and the distribution of PAHs in air, terrestrial, and aquatic environments?

- Effects of different environmental factors, such as pH, temperature, and nutrient availability, on each bioremediation approach for contaminants.

- Can you describe more about the intermediate products for each of the three bioremediation approaches?

- What is the effect of other contaminants, such as heavy metals, on the bioremediation approaches?

- Can you briefly describe other fungal bioremediation methods to compare with the three bioremediation methods described in your manuscript?

Author Response

We thank you the reviewer for taking the time to assess the manuscript. Below, we provide a point-by-point response. All revisions in the manuscript are highlighted.

 

Question 1 Can you describe more specifically the sources of PAHs and the distribution of PAHs in air, terrestrial, and aquatic environments?

Response 1 We agree. The PAHs section has been expanded from line 76 to 83.

 

 Question 2 Effects of different environmental factors, such as pH, temperature, and nutrient availability, on each bioremediation approach for contaminants.

Response 2 We found this comment highly relevant. The effects of pH, temperature and nutrient availability have been included in line 112-117 for biofiltration, line 230-240 in biosorption and in line 395-399 in biodegradation.

Question 3 Can you describe more about the intermediate products for each of the three bioremediation approaches?

Response 3Thank you for this valuable comment. We agree that describing the intermediate products formed during bioremediation is an important point. To address it more precisely, we would like to kindly ask whether your comment refers specifically to fungal-mediated biodegradation, or if you are suggesting that we should elaborate on the intermediates across all three bioremediation approaches in general.

 

Question 4 What is the effect of other contaminants, such as heavy metals, on the bioremediation approaches?

Response 4 Thank you for this suggestion. We agree that considering the effect of other contaminants, such as heavy metals, on bioremediation processes is indeed valuable. At the same time, we are mindful that this might broaden the scope of the paper beyond what we outlined in the introduction (lines 65–68).

Question 5 Can you briefly describe other fungal bioremediation methods to compare with the three bioremediation methods described in your manuscript?

Response 5 Thank you for this helpful comment. We agree that describing other fungal bioremediation methods can provide useful context for comparison. In the current manuscript, we have already introduced the concept of bioaccumulation within the section on biosorption (line 247), and we have also discussed biostimulation, bioaugmentation, biomineralization, and biotransformation within the biodegradation section (lines 398–417). While we recognize that these concepts could be elaborated further (for example, certain fungi can transform soluble pollutants such as metals into insoluble mineral forms, oxalates, sulfides or phosphates, thereby reducing their mobility and toxicity), we feel that expanding on them in greater detail may extend beyond the intended scope of this review.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper <Bioremediation of polycyclic aromatic hydrocarbons (PAHs) in aqueous environments: a review of biofiltration, biosorption and biodegradation strategies using living fungal mycelium>addresses a topic of major importance for the environment and human health as PAHs are pollutants widespread in various forms in the environment.

The manuscript is quite extensive and rich in useful theoretical and practical information, relatively up-to-date and particularly useful as a scientific information and awareness document. However, I recommend that the authors consider the following suggestions aimed at improving the scientific quality and relevance of this study.

1. I find it useful for the manuscript to begin with an Introduction section that could include Chapters 1 and 2 in their current form.
2. I ask authors to mention the originality of this study, what it brings new compared to other scientific review articles, what are the elements that have been less addressed in previous studies and that require detailed analysis and discussion.
3. I believe that sections 3, 4 and 5 could be merged into a single chapter addressing biofiltration of PAHs by fungi, summarizing and including less general theoretical information from sections 3 and 4.
4. Also, similar to the previous suggestion, I recommend that sections 6, 7 and 8 be merged into a single section addressing biosorption of PAHs by fungi.
5. Sections 9 and 11 should be merged into a single section on biodegradation of PAHs by fungi, without including section 10 on bacteria.
6. Discussions are quite brief compared to the detailed text, I recommend approaching this chapter in more depth.
7. Since the text contains many abbreviations, it would be useful to include a table of abbreviations.

Author Response

We sincerely thank the reviewer for their constructive feedback, which has significantly improved the clarity and contribution of our manuscript. Below, we provide a point-by-point response. All revisions in the manuscript are highlighted.

Question 1  I find it useful for the manuscript to begin with an Introduction section that could include Chapters 1 and 2 in their current form.

Response 1 We agree. We have restructured the introduction by including the first two sections of 1.1. Biological strategies for water remediation of PAHs: Biofiltration, Biosorption and Biodegradation and 1.2 PAHs.

Question 2 I ask authors to mention the originality of this study, what it brings new compared to other scientific review articles, what are the elements that have been less addressed in previous studies and that require detailed analysis and discussion.

Response 2 We agree with this comment and we have modified/expanded (line 9-14) in the abstract and  (line 53- 68) in the introduction, addressing on the innovation and boundary of this review.

 

Question 3 I believe that sections 3, 4 and 5 could be merged into a single chapter addressing biofiltration of PAHs by fungi, summarizing and including less general theoretical information from sections 3 and 4.

Response 3 Thank you for this constructive suggestion. We agree that a more concise structure can improve the readability of the manuscript. At the same time, we consider it important to provide a general introduction to contextualize the topic before addressing the specific case of PAH biofiltration by fungi. To improve clarity, we have revised the structure accordingly: Section 2 is now titled “Biofiltration and other methods”, with subsections 2.1 Overview of biofiltration, 2.2 Biofiltration of PAHs, and 2.3 Membrane filtration of PAHs through fungi. We believe this structure balances contextual information with a focused discussion of fungal biofiltration.

 

Question 4 Also, similar to the previous suggestion, I recommend that sections 6, 7 and 8 be merged into a single section addressing biosorption of PAHs by fungi.

Response 4 Similar to the previous answer we have introduce section 3 Biosorption and other methods  with subsections 3.1. Overview of biosorption, 3.2 Biosorption of PAHs and 3.3. Fungal biosorption of PAHs.

Question 5 Sections 9 and 11 should be merged into a single section on biodegradation of PAHs by fungi, without including section 10 on bacteria.

Response 5 Here we have introduced section 4 Biodegradation and other methods with subsections 4.1. Overview of biodegradation, 4.2. Biodegradation against PAHs through bacteria, 4.3. Fungal biodegradation against PAHs.

Question 6 Discussions are quite brief compared to the detailed text, I recommend approaching this chapter in more depth.

Response 6 We agree. Section 12 has been expanded (line 501- 517) with a design outline that incorporates influent water quality and the State of the art of concentration and types of PAHs effectively degraded. We have stated the constraints in a multi-solute scenario, optimal pH and temperature conditions, the most common fugal carriers, the importance of monitoring the water flux through the membrane and finally the potential of microbial partnership in remediation.

Question 7 Since the text contains many abbreviations, it would be useful to include a table of abbreviations.

Response 7 We agree there are several abbreviations relative to PAHs in the text. We have added those abbreviations in figure 4 pag 6 for each of the 16 PAHs.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The issues in the manuscript have been well addressed, and the paper can be accepted after minor revisions. The specific details of the references are as follows:"Microalgal biofilm cultivation on lignocellulosic based bio-carriers: Effects of material physical characteristics on microalgal biomass production and composition, Chemical Engineering Journal,Volume 510,2025,161656, https://doi.org/10.1016/j.cej.2025.161656."

Author Response

Thank you for redirecting us to the right link.

We have included the reference and explanation, it is highlighted 120-124.

Author Response File: Author Response.pdf