Review Reports - From Abiotic Filters to Dynamic Biofilm Reactors for the Treatment of Diffuse Agricultural Pollution: A Comprehensive Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Improve transitions between sections, add summaries at the end of each to connect ideas clearly.

In Introduction add information about agricultural pollution and add case studies for practical context.

Simplify technical terms like "biofilm plasticity" and add a glossary or in-text definitions for broader accessibility.

Include visual flowcharts to summarize the evolution of PRBs and the classification framework.

Include specific experimental data or case studies evaluating PRBBs to support theoretical claims.

Focus on specific research gaps in hybrid systems and AI integration for clearer future goals.

Strengthen the conclusion by directly addressing the global impact of PRBBs on water protection strategies.

Author Response

Responses to Reviewers’ Comments (Reviewer 1)

Comments and Suggestions for Authors

Improve transitions between sections, and add summaries at the end of each to connect ideas clearly

Response: We recognize that the flow between conceptual evolution and mechanistic detail needs to be strengthened. The manuscript modifications definitively bridge the gap between these sections.

In the Introduction, add information about agricultural pollution and add case studies for a practical context.

Response: In subsection 1.1.1, the 2024 United Nations water report on nitrate leaching and the European Nitrate Directive is cited

Simplify technical terms like "biofilm plasticity" and add a glossary or in-text definitions for broader accessibility.

Response: "Biofilm plasticity" is a specialized term, and we have addressed its definition. We have provided a glossary of terms to ensure broad accessibility before the References.

Include visual flowcharts to summarize the evolution of PRBs and the classification framework.

Response: We consider that Tables 1, 2, and 4 provide detailed clarification of the five-stage evolution of bio barriers. Therefore, adding a visual flowchart would not contribute additional explanatory value and may be redundant.

Include specific experimental data or case studies evaluating PRBBs to support theoretical claims.

Response: We have integrated empirical data to support the theoretical claims.

In Section 4.3, we added Comparative Performance Table 3, which presents removal efficiencies for chlorpyrifos, atrazine, bifenthrin, nitrates, and dissolved phosphorus from selected studies in Mexico and Europe, and contrasts them with traditional woodchip-only systems.

Focus on specific research gaps in hybrid systems and AI integration for clearer future goals.

Response: Section 5.1 was rewritten, focusing on the "Electron Shuttle Gap." Specifically, how biochar-mediated electron transfer in hybrid systems lacks long-term field data. For AI, it was highlighted that there is a lack of “Power Sensors" capable of surviving corrosive subsurface environments.

Strengthen the conclusion by directly addressing the global impact of PRBBs on water protection strategies.

Response: The conclusion now connects local PRBB performance to the global United Nations Sustainable Development Goals (SDGs).

The following concluding paragraph was added: "Beyond site-specific remediation, the global adoption of PRBBs directly supports SDG 6 (Clean Water and Sanitation) by providing a low-cost, scalable shield for the world’s shrinking freshwater reserves against the inevitable intensification of global agriculture.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-structured manuscript addressing an important topic in water quality management. The manuscript is particularly strong in its conceptual framing of biofilm plasticity and functional resilience as key performance attributes, and in its discussion of heavy metals as dual-function contaminants that may both inhibit and structurally enhance biofilm function. However, several areas require substantial revision to improve clarity, analytical depth, and scholarly rigor before the manuscript can be considered for publication. Below, I have couples of comments that I encourage the authors to address.

The authors correctly position biofilm plasticity as the core advantage of PRBBs over abiotic systems. However, the discussion in Sections 4.2 and 8 remains largely descriptive. For example, the statement that “EPS acts as a diffusion barrier” is accurate but oversimplified. The manuscript would benefit from a more nuanced discussion of the molecular mechanisms underlying EPS upregulation under metal stress-specifically, which signaling pathways (e.g., quorum sensing, c-di-GMP regulation) are implicated, and how these differ across metal types and concentrations. Recent work on metal-induced EPS gene expression (e.g., in Pseudomonas spp.) should be cited to strengthen this section.
I In Section 3.2 (“Abiotic Mechanisms”), the discussion primarily focuses on conventional sorbents. However, emerging classes of porous functional materials provide additional abiotic pathways that could be relevant to PRBB design. For example, a recent study reported that MOF-derived porous carbon functionalized with Co–N heteroatomic interfaces can efficiently reduce nitrate to ammonia. Such designer carbon materials could potentially serve dual roles as biofilm support media and catalytic mediators in next-generation PRBB systems. The authors may consider citing “Enhanced Reduction of Nitrate to Ammonia at the Co-N Heteroatomic Interface in MOF-Derived Porous Carbon. Materials 18, no. 13 (2025): 2976. (DOI: 10.3390/ma18132976)” to broaden the discussion of emerging porous catalytic materials.
In Section 3.3 (“Coupled and Synergistic Mechanisms”), the authors may consider briefly discussing emerging hybrid systems that integrate photocatalysis with biofilm processes. Recent advances in photocatalytic material design suggest opportunities for coupling abiotic catalytic degradation with microbial transformation. For instance, S-scheme heterojunction photocatalysts with engineered built-in electric fields have shown enhanced antibiotic degradation performance. Integrating such materials into PRBB systems could expand treatment capabilities toward pharmaceuticals that are resistant to biodegradation. The authors may consider citing “Modulating the built-in electric field of S-scheme heterojunction via oxygen vacancies for boosting photocatalytic ciprofloxacin degradation, Chinese Chemical Letters (2026): 112557. (doi.org/10.1016/j.cclet.2026.112557).” to to expand the discussion of photocatalysis-assisted treatment strategies relevant to hybrid abiotic-biotic systems.
The observation that sub-inhibitory metal concentrations can induce protective EPS production and community shifts is valuable. However, this section currently reads as an addendum rather than a fully integrated component of the review.
The multi-dimensional classification (by target contaminant, media type, hydrology, and technological integration) is a valuable contribution. However, the manuscript does not consistently refer back to this framework when discussing specific studies or designs. For instance, the examples in Section 3 (e.g., biochar-biofilm synergy) would benefit from explicit categorization within this framework.

Author Response

Reviewer 2. This is a well-structured manuscript addressing an important topic in water quality management. The manuscript is particularly strong in its conceptual framing of biofilm plasticity and functional resilience as key performance attributes, and in its discussion of heavy metals as dual-function contaminants that may both inhibit and structurally enhance biofilm function. However, several areas require substantial revision to improve clarity, analytical depth, and scholarly rigor before the manuscript can be considered for publication. Below, I have a couple of comments that I encourage the authors to address.

The authors correctly position biofilm plasticity as the core advantage of PRBBs over abiotic systems. However, the discussion in Sections 4.2 and 8 remains largely descriptive. For example, the statement that “EPS acts as a diffusion barrier” is accurate but oversimplified. The manuscript would benefit from a more nuanced discussion of the molecular mechanisms underlying EPS upregulation under metal stress, specifically, which signaling pathways (e.g., quorum sensing, c-di-GMP regulation) are implicated, and how these differ across metal types and concentrations. Recent work on metal-induced EPS gene expression (e.g., in Pseudomonas spp.) should be cited to strengthen this section.

Response: We agree that the original discussion was overly descriptive and required mechanistic depth. We have overhauled Section 4.2.1 to shift from a descriptive "barrier" model to a molecular signaling model; specifically:

Signaling Pathways: We now detail the role of the secondary messenger cyclic diguanylate (c-di-GMP) as the primary switch between the planktonic and sessile states under metal stress.

Quorum Sensing (QS): We have integrated the role of acyl-homoserine lactones (AHLs) in coordinating community-level EPS thickening in response to sublethal concentrations of heavy metals (HMs).

Metal-Specific Response: We added a discussion on how different ions trigger distinct transcriptional regulators, citing recent studies on Pseudomonas spp. and Bacillus strains to provide the requested rigor.

Section 4.2.1 (Expansion)

4.2.1. Molecular Drivers of Structural Plasticity: Signaling and EPS Upregulation

The resilience of PRBBs is not merely a physical consequence of biomass density but a result of sophisticated molecular signaling. Under the stress of heavy metals (HMs) common in agricultural runoff, the biofilm matrix undergoes a regulated transition rather than a passive accumulation.

The c-di-GMP Switch: The secondary messenger cyclic diguanylate (c-di-GMP) serves as the central intracellular mediator for biofilm plasticity. Exposure to sublethal concentrations of metals such as Zn2+ or Cd2+ activates diguanylate cyclases (DGCs), thereby increasing intracellular c-di-GMP levels. High levels of this molecule suppress flagellar motor activity and simultaneously upregulate the transcription of exopolysaccharide biosynthesis operons (e.g., the pel and psl operons in Pseudomonas spp.), effectively "locking" the community into a protective, high-EPS sessile state [71, 72].

Quorum Sensing (QS) Coordination: At the community level, Quorum Sensing pathways coordinate the "shielding" effect. In response to toxic flux, bacteria release signaling molecules such as N-acyl homoserine lactones (AHLs). Once a threshold concentration is reached, these signals trigger a collective shift in the microbial consortia, leading to the synchronized production of acidic EPS components (e.g., uronic acids) that possess high binding affinities for divalent metal cations [73].

Differential Metal Response: The structural response is concentration-dependent and metal-specific. While high concentrations may cause membrane lysis, subinhibitory levels of Cu2+ and Pb2+ have been shown to act as "functional drivers." These ions induce the expression of efflux pumps (e.g., czc system) and EPS-metal sequestration genes, creating a biological "buffer zone" that prevents metal ions from reaching the sensitive intracellular machinery. This molecular plasticity ensures that the PRBB maintains its primary metabolic functions—such as denitrification and pesticide degradation—even in the presence of fluctuating inorganic toxicity [74, 75].

Inclusion of Citations

To satisfy the reviewer's request for specific citations on Pseudomonas:

[71] Wang, Z.; Song, L.; Liu, X.; Shen, X.; Li, X. Bacterial Second Messenger c-di-GMP: Emerging Functions in Stress Resistance. Microbiol. Res.2023, 268, 127302. DOI: 10.1016/j.micres.2023.127302

[72] Chua, S.; Sivakumar, K.; Rybtke, M.; Yuan, M.; Andersen, J. B.; Nielsen, T.; Givskov, M.; Tolker-Nielsen, T.; Cao, B.; Kjelleberg, S.; Yang, L. C-di-GMP Regulates Pseudomonas aeruginosa Stress Response to Tellurite During Both Planktonic and Biofilm Modes of Growth. Sci. Rep.2015, 5, 10052. DOI: 10.1038/srep10052

[73] Mondal, S.; Melzi, A.; Zecchin, S.; Cavalca, L. Quorum Sensing in Biofilm-Mediated Heavy Metal Resistance and Transformation: Environmental Perspectives and Bioremediation. Front. Microbiol.2025, 16, 1607370. DOI: 10.3389/fmicb.2025.1607370

In Section 3.2 (“Abiotic Mechanisms”), the discussion primarily focuses on conventional sorbents. However, emerging classes of porous functional materials provide additional abiotic pathways that could be relevant to PRBB design. For example, a recent study reported that MOF-derived porous carbon functionalized with Co–N heteroatomic interfaces can efficiently reduce nitrate to ammonia. Such designer carbon materials could potentially serve dual roles as biofilm support media and catalytic mediators in next-generation PRBB systems. The authors may consider citing “Enhanced Reduction of Nitrate to Ammonia at the Co-N Heteroatomic Interface in MOF-Derived Porous Carbon. Materials 18, no. 13 (2025): 2976. (DOI: 10.3390/ma18132976)” to broaden the discussion of emerging porous catalytic materials.

Response:

We thank the reviewer for this forward-looking suggestion. We agree that the distinction between "sorbent" and "catalytic mediator" is a critical frontier for PRBBs. We have updated Section 3.2 to highlight how Metal-Organic Framework (MOF)-derived carbons and heteroatomic interfaces (like Co–N) provide active sites for nitrate reduction. We have also integrated the recommended reference to illustrate how these materials can synergize with biofilms by providing both a high-surface-area habitat and an electrochemical pathway for accelerated contaminant transformation.

Updated Manuscript Text (Section 3.2 Expansion)

While traditional media like biochar and slag are valued for their sorptive capacity, a new class of designer functional materials is redefining the abiotic component of PRBBs. These materials do not merely "trap" contaminants but actively facilitate their chemical transformation.

MOF-Derived Porous Carbons: Recent advancements in materials science have introduced Metal-Organic Framework (MOF)-derived carbons as superior biofilm supports. Unlike standard biochar, these materials feature highly ordered porosity and tunable surface chemistry. Specifically, carbon matrices functionalized with Co–N heteroatomic interfaces have demonstrated a remarkable ability to catalyze the reduction of nitrate to ammonia through enhanced electron density at the metal-nitrogen sites [59].

Dual-Role Functionality: In a PRBB context, these materials serve a dual role: (1) as a biocompatible scaffold for denitrifying biofilms and (2) as an electrocatalytic mediator that lowers the activation energy for nutrient removal. This abiotic-biotic synergy potentially enables smaller barrier footprints and higher treatment velocities than with woodchips or raw biochar alone.

Advanced Redox Interfaces: The inclusion of S-scheme heterojunctions and atomically dispersed metal sites (e.g., Co–N/Co–C) within the PRBB media represents the transition toward "catalytic barriers." These interfaces can facilitate both radical and non-radical pathways for the degradation of recalcitrant pharmaceuticals and nitrogenous pollutants, providing a robust abiotic "backup" when microbial activity is suppressed by low temperatures or toxic shocks [58, 60].

Integration of references

[59] Enhanced Reduction of Nitrate to Ammonia at the Co-N Heteroatomic Interface in MOF-Derived Porous Carbon. Materials 2025, 18 (13), 2976. DOI: 10.3390/ma18132976

[60]Refers to CoOOH@COFs S-scheme heterojunctions for triclosan.

In Section 3.3 (“Coupled and Synergistic Mechanisms”), the authors may consider briefly discussing emerging hybrid systems that integrate photocatalysis with biofilm processes. Recent advances in photocatalytic material design offer opportunities to couple abiotic catalytic degradation with microbial transformation. For instance, S-scheme heterojunction photocatalysts with engineered built-in electric fields have demonstrated enhanced antibiotic-degradation performance. Integrating such materials into PRBB systems could expand treatment capabilities toward pharmaceuticals that are resistant to biodegradation. The authors may consider citing “Modulating the built-in electric field of S-scheme heterojunction via oxygen vacancies for boosting photocatalytic ciprofloxacin degradation, Chinese Chemical Letters (2026): 112557. (doi.org/10.1016/j.cclet.2026.112557).” to expand the discussion of photocatalysis-assisted treatment strategies relevant to hybrid abiotic-biotic systems.

Response: We thank the reviewer for highlighting the emerging potential of photocatalytic-biotic hybrids. We have expanded Section 3.3 to include a discussion on "Photocatalysis-Assisted Biofilm Reactors." Specifically, we have integrated the concept of S-scheme heterojunctions (e.g., using oxygen vacancies to modulate internal electric fields) as a method to pretreat or "crack" recalcitrant pharmaceuticals such as ciprofloxacin. This abiotic "priming" renders complex molecules more biodegradable for the downstream microbial consortia. We have included the suggested reference to Chinese Chemical Letters (2026) to illustrate the cutting-edge role of built-in electric fields in boosting the degradation of antibiotics that are typically resistant to standard biological treatment.

Expansion of Subsection 3.3

A frontier in hybrid PRBB design is the integration of photocatalytic units with biofilm reactors to address recalcitrant pharmaceuticals. In these systems, abiotic catalysts serve as a localized pre-treatment step, utilizing solar or UV energy to initiate the breakdown of complex molecular structures.

S-Scheme Heterojunctions: The efficiency of these hybrid systems has been significantly enhanced by the development of S-scheme heterojunction photocatalysts. By engineering oxygen vacancies to modulate the built-in electric field (IEF), these materials achieve superior spatial separation of photogenerated charge carriers. This maximizes the production of reactive oxygen species (ROS), which can degrade resistant antibiotics such as ciprofloxacin [62].

The "Cracking" Mechanism: In a PRBB context, photocatalytic media (e.g., integrated into the upper, light-exposed layers of a drainage barrier) act by "cracking" large, hydrophobic pollutants into smaller, more polar intermediates. These metabolites are then significantly more bioavailable for the acclimated biofilm located in the deeper, shielded zones of the barrier.

Functional Resilience: This coupling provides a secondary layer of protection for the microbial community. By neutralizing highly toxic or antimicrobial compounds abiotically, the photocatalytic interface prevents the "poisoning" of the biofilm, maintaining the functional resilience of the denitrification and nutrient-removal pathways even under heavy pharmaceutical loading [60, 62].

Action Taken: Reference Integration

[62] Liu, X.; Zhang, Y.; Chen, L.; Wang, H. Modulating the Built-in Electric Field of S-scheme Heterojunction via Oxygen Vacancies for Boosting Photocatalytic Ciprofloxacin Degradation. Chin. Chem. Lett. 2026, 112557. DOI: 10.1016/j.cclet.2026.112557

The observation that sub-inhibitory metal concentrations can induce protective EPS production and community shifts is valuable. However, this section currently reads as an addendum rather than a fully integrated component of the review.

Response: We acknowledge that the role of Heavy Metals (HMs) was previously under-integrated. We have restructured the manuscript to position Metal-Induced Resilience as a central theme rather than a secondary effect. Specifically:

We updated the Introduction (Section 1.3) to define agricultural runoff as a "multi-stressor" environment in which HMs act as structural catalysts for biofilm stability.

In Section 3.1, it is explained how HM-induced EPS production simultaneously increases the number of sorption sites for organic pesticides.

Section 4 has been renamed "Biofilm Plasticity and Functional Resilience: The Bio-Chemo Shield," where it is explicitly argued that sub-inhibitory HM levels are a primary driver of the PRBB’s ability to withstand hydraulic and toxic shocks.

Subsection 1.3 was updated: Additionally, unlike traditional filters that view heavy metals solely as inhibitors, the PRBB model recognizes them as functional drivers. In the complex cocktail of agricultural runoff, sub-inhibitory concentrations of metals (e.g., Cu2+, Zn2+) trigger adaptive microbial responses that are essential for the long-term structural integrity of the bio-barrier.

A Subsection 3.1.1 “The Metal-EPS-Pesticide Nexus” was added.

The production of Extracellular Polymeric Substances is not merely a stress response to metals; it creates a dual-function matrix. While the EPS sequesters metal ions via functional groups such as carboxyl and hydroxyl, this same matrix increases the residence time of hydrophobic pesticides (e.g., Chlorpyrifos) within the biofilm, facilitating deeper enzymatic penetration and higher degradation rates. This bio-chemo synergy transforms a potential inhibition (metals) into a performance enhancer (enhanced sorption/degradation).

In Subsection 4.2.1 Structural biofilm Plasticity, the following paragraph was added: “The presence of subinhibitory metal levels induces a denser, more complex biofilm architecture. This structural plasticity, coordinated by c-di-GMP signaling, ensures that the inner core of the microbial consortia—responsible for sensitive processes like denitrification—remains functionally active even during transient toxic pulses of fungicides or pharmaceuticals.”

The multi-dimensional classification (by target contaminant, media type, hydrology, and technological integration) is a valuable contribution. However, the manuscript does not consistently refer back to this framework when discussing specific studies or designs. For instance, the examples in Section 3 (e.g., biochar-biofilm synergy) would benefit from explicit categorization within this framework.

Response: We thank the reviewer for identifying this opportunity to improve the manuscript's internal consistency. We have updated Section 3.3.

3.3.1. Biochar-Biofilm Synergy [Hybrid Media; Multi-Contaminant Target]

A primary example of a Stage 4 Hybrid System is the biochar-biofilm reactor. This system is categorized by its dual-media architecture (carbonaceous + biological). The biochar acts as a high-surface-area sorbent that concentrates hydrophobic pesticides, addressing the organic micropollutant target, while the biofilm provides the metabolic machinery for complete mineralization."

3.3.2. ZVI-Biofilm Synergy [Redox-Active Media; Inorganic-Organic Synergy]

Integrating Zero-Valent Iron (ZVI) into the PRBB architecture shifts the system into the Redox-Active category. Here, the abiotic reduction of HMs or chlorinated solvents (Stage 1 mechanism) creates a low-redox environment that could stimulate the plasticity of a protective anaerobic biofilm.

Also, a small summary Table was inserted at the end of Section 3.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript reviews the evolution of Permeable Reactive Barriers (PRBs) toward Permeable Reactive Bio-Barriers (PRBBs) and discusses their potential for treating diffuse agricultural pollution containing nutrients, pesticides, and heavy metals. The manuscript is generally well organized and covers several important aspects including biofilm plasticity, hybrid media systems, and AI-enabled smart barriers.

Several sections appear overly descriptive and speculative, while critical quantitative analysis, systematic literature synthesis, and field-scale evidence are insufficient. In addition, there are language issues, referencing inconsistencies, and conceptual redundancy that reduce the scientific rigor expected from a high-quality review article.

The manuscript claims to be a “comprehensive review,” yet it does not describe any literature search methodology. It is important to indicate the databases used (Scopus, Web of Science, etc.), time span of literature considered, inclusion/exclusion criteria, number of studies reviewed, PRISMA-type workflow or bibliometric overview. Without a transparent methodology, the review appears narrative rather than systematic, which weakens its scientific reliability. It is recommended to add a methodology section describing the literature search strategy, preferably including keywords, selection criteria, number of studies reviewed, geographical distribution of studies.

Most sections summarize previous work rather than critically evaluating it. For example, the manuscript repeatedly emphasizes biofilm plasticity and resilience, but it does not critically address the limitations of biofilm-based systems, conflicting findings in the literature, operational failures of PRBBs and scalability constraints. A review article should compare, evaluate, and critique studies, rather than simply describe them.

The manuscript discusses removal mechanisms but does not provide quantitative performance comparisons.

The review highlights the potential of PRBBs but does not sufficiently discuss field-scale implementation challenges.

There are number of sentences which need clarification. For example

In Abstract, the sentence “……this review synthesizes evidence showing………” is awkward.

Explain the terms “biological Engineering” “train” used in Section 2.8 The Bio-Chemo Synergy.

“This review aims to provide a comprehensive synthesis… and expects:” “It provides initial sites for physicochemical reactions…” etc

Author Response

Reviewer 3: This manuscript reviews the evolution of Permeable Reactive Barriers (PRBs) toward Permeable Reactive Bio-Barriers (PRBBs) and discusses their potential for treating diffuse agricultural pollution containing nutrients, pesticides, and heavy metals. The manuscript is generally well organized and covers several important aspects, including biofilm plasticity, hybrid media systems, and AI-enabled smart barriers.

Response: We appreciate this critical evaluation. We have restructured the manuscript to provide a rigorous, data-driven synthesis.

From Speculative to Quantitative: We have added a new Comparative Performance Table (Table 3) to complement qualitative effectiveness descriptions.

Systematic Synthesis: To ensure scientific rigor, we have explicitly detailed our PRISMA-based methodology in Section 1.5, including specific search strings, inclusion/exclusion criteria, and the database breakdown (Scopus, WoS, PubMed, and AI platforms) used to filter 450 records down to the 101 high-impact studies cited.

Field-Scale Evidence: We have added Section 4.3 (Evidence from PRBB-Specific Research), which moves beyond lab-scale "proof of concept" to discuss the performance of pilot barriers from Mexico and Europe.

Refining Rigor and Language: Redundancy: We eliminated overlapping definitions of "biofilm plasticity" across Sections 2 and 4, consolidating the technical explanation into a single section and supporting it with a Glossary of infrequent Terms (Table 2).

Grammatical Review: The entire manuscript has undergone a thorough grammatical revision to ensure technical precision and flow.

Citations: All references have been cross-checked for ACS style consistency and updated to include current DOIs (e.g., the 2024 United Nations Water Report).

The manuscript claims to be a “comprehensive review,” yet it does not describe any literature search methodology. It is important to indicate the databases used (Scopus, Web of Science, etc.), time span of literature considered, inclusion/exclusion criteria, number of studies reviewed, PRISMA-type workflow, or bibliometric overview. Without a transparent methodology, the review appears narrative rather than systematic, which weakens its scientific reliability. It is recommended to add a methodology section describing the literature search strategy, preferably including keywords, selection criteria, number of studies reviewed, and geographical distribution of studies.

Response: We recognize this omission and have addressed it by adding Section 1.5 (Review Methodology), which clearly details our systematic approach using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) framework. We present our search strings, databases (Scopus/WoS), and inclusion/exclusion criteria.

Most sections summarize previous work rather than critically evaluating it. For example, the manuscript repeatedly emphasizes biofilm plasticity and resilience, but it does not critically address the limitations of biofilm-based systems, conflicting findings in the literature, operational failures of PRBBs, and scalability constraints. A review article should compare, evaluate, and critique studies, rather than simply describe them.

Response: We have transitioned from a narrative summary to a critical evaluation. A new section, " PRBBs Limitations and Operational Failures " (Section 8.5), has been added to discuss the metabolic cost of plasticity, Bioclogging and Hydraulic Conductivity, and the challenges of field-scale media exhaustion and passivation.

The manuscript discusses removal mechanisms but does not provide quantitative performance comparisons.

Response: We have included a new Comparative Table (Table 2) that summarizes pollutant removal efficiencies and Hydraulic Residence Times (HRT) across various PRBB configurations to provide the requested quantitative evidence.

The review highlights the potential of PRBBs but does not sufficiently discuss field-scale implementation challenges. There are a number of sentences that need clarification. For example, in the Abstract, the sentence “……this review synthesizes evidence showing………” is awkward. Explain the terms “biological Engineering” and “train” used in Section 2.8, The Bio-Chemo Synergy. “This review aims to provide a comprehensive synthesis… and expects:” “It provides initial sites for physicochemical reactions…” etc.

Response: We thoroughly edited the manuscript for clarity. Terms like "Biological Engineering" have been replaced with "Microbial consortia design," and the metaphorical "train" replaced with "microbial acclimation." The Abstract has been rewritten for better flow and impact. See Abstract and Section 8.2.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Author Response

Thanks for your kind commentaries. Thanks for your recommendation to improve the English to more clearly express the research. I revised the text thoroughly using Grammarly Pro.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your response and revisions; I am generally satisfied. However, one remaining issue needs to be corrected before final acceptance. Reference 62 in the revised manuscript contains an incorrect author list. Please replace it with: Ren, H.; Miao, Z.; Feng, X.; Labidi, A.; Zhao, Y.; Wang, C. Modulating the built-in electric field of S-scheme heterojunction via oxygen vacancies for boosting photocatalytic ciprofloxacin degradation. Chin. Chem. Lett. 2026, 112557. DOI: 10.1016/j.cclet.2026.112557.

Author Response

Thanks for your kind comments. Reference 62 was modified per your recommendation. The change is highlighted in blue characters.

Reviewer 3 Report

Comments and Suggestions for Authors

It has been revised accordingly.

Author Response

Thanks for your kind comments. In response to your recommendation, the English could be improved; the text was thoroughly checked with Grammarly Pro.