Bioremediation of Synthetic Dyes by White-Rot Fungi: Enzymatic Mechanisms, Biosorption, and Environmental Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the article Manuscript ID: molecules-4198408

Title: Bioremediation of Synthetic Dyes by White Rot Fungi: Enzymatic

Mechanisms, Biosorption, and Environmental Applications

 

This article examines the pressing issue of bioremediation of synthetic dyes using white rot fungi. This topic is important for modern biotechnology, which aims to employ environmentally friendly and safe technologies, including those using various types of macromycetes.

The widespread use of dyes in the textile industry leads to contamination of aquatic ecosystems with persistent and toxic compounds that are difficult to remove using traditional methods.

This manuscript presents current data on the enzymatic mechanisms of biodegradation of synthetic dyes by white-rot fungi, analyzes existing biotechnological approaches, and explores the prospects for their implementation in environmentally friendly treatment technologies.

In this regard, the use of white-rot fungi and their ligninolytic enzymes, capable of effectively breaking down the complex aromatic structures of dyes, is of particular interest.

A review of current research in this area is essential for the development of environmentally friendly and efficient wastewater treatment technologies.

The authors demonstrate a deep understanding of the biochemistry of ligninolytic enzymes. Key macromycete species producing these enzymes, such as Pleurotus species, Bjerkandera adusta, Trametes versicolor, Phlebia radiata, and Phanerochaete chrysosporium, are discussed in detail. The mechanisms of action of lignin peroxidase, manganese peroxidase, dye-decolorizing peroxidase, and laccase are described. The processes of biosorption, bioaccumulation, and mineralization of dyes are discussed. Examples of industrial wastewater bioremediation are provided.

Of particular importance is a detailed description of the enzyme catalytic cycles and their structural features, including the involvement of metal ions and cofactors (e.g., heme groups, Cu²⁺, and flavin adenine dinucleotide).

The article presents informative diagrams and illustrations to facilitate understanding of the mechanisms of enzymatic degradation.

The reference list includes current and relevant sources (2020–2025), including articles from leading journals. Furthermore, the authors consulted modern scientific publications on enzymology, bioremediation, and fungal biotechnology.

The article is fully consistent with the scope of the journal Molecules, as it focuses on the enzymatic mechanisms of synthetic dye biodegradation and the biotechnological application of ligninolytic enzymes from white rot fungi, which fall within the fields of molecular biotechnology, enzymology, and environmental chemistry.

The following comments are intended to enhance the scientific clarity and methodological completeness of the review:

Despite the overall high rating, I suggest the authors address the following points to improve the manuscript:

1. In the introduction, it is advisable to more clearly formulate the scientific novelty and main goals of the review, as well as highlight the differences between this work and previously published review articles on this topic.
2. The caption for Figure 2 should be updated to include full attribution for the photographs. Please specify the authors/photographers of the images and indicate the location where the fungal specimens were collected or photographed. This information is necessary to ensure proper scientific documentation and compliance with publication standards. If the authors took the photographs, the caption should explicitly state “Photos by the authors,” and the collection site (e.g., country, region, or habitat) should also be indicated. If the images were reproduced or adapted from other sources, the original references and permissions must be clearly provided.
3. Section 7. "Problems and Prospects" could be supplemented with a brief mention of existing approaches aimed at overcoming the limitations noted by the authors. For example, enzyme immobilization strategies or improved bioreactor designs, overexpression of ligninolytic enzymes, and metabolic engineering to enhance degradation capacity. Adding a few suggestions in these areas would enhance the discussion.
4. The concluding section clearly summarizes the review's main points and highlights the importance of white-rot fungi in the biodegradation of synthetic dyes.

However, this section could be improved:

The conclusion could more clearly summarize the key scientific findings of the review, such as the comparative effectiveness of the main ligninolytic enzymes or the most promising fungal species for dye degradation.

Also, it would be useful to briefly highlight the most promising technological approaches, such as enzyme immobilization, the use of fungal consortia, or combined treatment systems combining biological and physicochemical methods.

Overall assessment and recommendation

Overall, the article is a relevant, scientifically sound, and practically relevant review. It is well-structured, analytical, and covers contemporary literature.

Recommended for publication after minor editorial revisions, taking into account the comments made.

 

Author Response

Response to reviewers’ comments:

 

Reviewer 01:

This article examines the pressing issue of bioremediation of synthetic dyes using white rot fungi. This topic is important for modern biotechnology, which aims to employ environmentally friendly and safe technologies, including those using various types of macromycetes. The widespread use of dyes in the textile industry leads to contamination of aquatic ecosystems with persistent and toxic compounds that are difficult to remove using traditional methods. This manuscript presents current data on the enzymatic mechanisms of biodegradation of synthetic dyes by white-rot fungi, analyzes existing biotechnological approaches, and explores the prospects for their implementation in environmentally friendly treatment technologies. In this regard, the use of white-rot fungi and their ligninolytic enzymes, capable of effectively breaking down the complex aromatic structures of dyes, is of particular interest. A review of current research in this area is essential for the development of environmentally friendly and efficient wastewater treatment technologies.

The authors demonstrate a deep understanding of the biochemistry of ligninolytic enzymes. Key macromycete species producing these enzymes, such as Pleurotus species, Bjerkandera adusta, Trametes versicolor, Phlebia radiata, and Phanerochaete chrysosporium, are discussed in detail. The mechanisms of action of lignin peroxidase, manganese peroxidase, dye-decolorizing peroxidase, and laccase are described. The processes of biosorption, bioaccumulation, and mineralization of dyes are discussed. Examples of industrial wastewater bioremediation are provided. Of particular importance is a detailed description of the enzyme catalytic cycles and their structural features, including the involvement of metal ions and cofactors (e.g., heme groups, Cu²⁺, and flavin adenine dinucleotide).

The article presents informative diagrams and illustrations to facilitate understanding of the mechanisms of enzymatic degradation. The reference list includes current and relevant sources (2020–2025), including articles from leading journals. Furthermore, the authors consulted modern scientific publications on enzymology, bioremediation, and fungal biotechnology. The article is fully consistent with the scope of the journal Molecules, as it focuses on the enzymatic mechanisms of synthetic dye biodegradation and the biotechnological application of ligninolytic enzymes from white rot fungi, which fall within the fields of molecular biotechnology, enzymology, and environmental chemistry.

R.: We thank the reviewer for the careful reading of our manuscript and for the positive evaluation of its scientific content and relevance. The comments and suggestions provided were very valuable and have been carefully considered in the revision of the manuscript, contributing to improving its clarity and overall quality.

 

The following comments are intended to enhance the scientific clarity and methodological completeness of the review:

 

Despite the overall high rating, I suggest the authors address the following points to improve the manuscript:

In the introduction, it is advisable to more clearly formulate the scientific novelty and main goals of the review, as well as highlight the differences between this work and previously published review articles on this topic.

R.: Thank you for this valuable suggestion. The introduction was revised to more clearly highlight the scientific novelty and the main objectives of this review. Additionally, a paragraph was added to emphasize the differences between this review and previously published review articles on fungal degradation of synthetic dyes.

 

Lines 106-125: In this context, the present literature review aims to analyze and systematize the use of WRF in the bioremediation of synthetic industrial dyes, with emphasis on the mechanisms involved, the main ligninolytic enzymes, and the conditions that influence process efficiency. Several review studies have previously addressed the application of fungi in dye removal, however, many of them focus mainly on specific enzymatic systems, individual fungal species, or general bioremediation approaches. Despite the growing number of studies on this topic, the literature still presents gaps related to the comparative evaluation of different white-rot fungal species and the translation of results obtained at laboratory scale to industrial scale applications. Furthermore, many studies address biochemical or environmental aspects in isolation, without a critical integration between enzymatic mechanisms and the technological potential of bioremediation. Therefore, a comprehensive analysis that integrates enzymatic mechanisms, fungal diversity, and operational conditions is still required to better understand the potential of WRF for the treatment of dye-contaminated effluents. In this sense, this review seeks to contribute to the advancement of the knowledge frontier by providing an integrated overview of recent advances in the use of WRF for the bioremediation of synthetic dyes, identifying current limitations, and highlighting perspectives for the development of more efficient, sustainable, and scalable processes for the treatment of industrial effluents containing synthetic dyes.

The caption for Figure 2 should be updated to include full attribution for the photographs. Please specify the authors/photographers of the images and indicate the location where the fungal specimens were collected or photographed. This information is necessary to ensure proper scientific documentation and compliance with publication standards. If the authors took the photographs, the caption should explicitly state “Photos by the authors,” and the collection site (e.g., country, region, or habitat) should also be indicated. If the images were reproduced or adapted from other sources, the original references and permissions must be clearly provided.

R.: Thank you for this important observation. The images presented in Figure 2 are illustrative representations generated using the program Gemini based on morphological characteristics of the fungal taxa described in the literature.

 

Section 7. "Problems and Prospects" could be supplemented with a brief mention of existing approaches aimed at overcoming the limitations noted by the authors. For example, enzyme immobilization strategies or improved bioreactor designs, overexpression of ligninolytic enzymes, and metabolic engineering to enhance degradation capacity. Adding a few suggestions in these areas would enhance the discussion.

R.: Thank you for this valuable suggestion. We have expanded Section 7 to briefly discuss strategies that may help overcome the limitations associated with fungal dye degradation systems.

 

Lines 667-690: 5. Strategies to overcome current limitations:

Recent advances in biotechnology and bioprocess engineering have provided promising strategies to overcome several limitations associated with fungal bioremediation systems. One of the most widely explored approaches is enzyme immobilization, which can significantly enhance enzyme stability, reusability, and resistance to adverse environmental conditions. Immobilized ligninolytic enzymes, such as laccases and peroxidases, have demonstrated improved catalytic performance and operational stability when applied to dye degradation processes (Ortolan et al., 2024; Cheute et al., 2025; Uber et al., 2025).

Another important strategy involves the development of improved bioreactor configurations designed to optimize fungal growth and enzymatic activity. Reactor systems such as packed-bed reactors, fluidized-bed reactors, and membrane bioreactors have been investigated to enhance mass transfer, maintain favorable environmental conditions, and increase the efficiency of pollutant removal in continuous treatment processes (Gomes et al., 2023).

In addition, advances in molecular biology and fungal biotechnology have opened new opportunities for improving the degradation capacity of fungal systems. Genetic engineering and heterologous expression of ligninolytic enzymes have been explored to increase enzyme production and catalytic efficiency (Contato et al., 2025). Similarly, metabolic engineering approaches may enable the development of fungal strains with enhanced tolerance to toxic compounds and improved degradation pathways (Salazar-Cerezo et al., 2023; Garg et al., 2025).

Together, these strategies highlight the potential for integrating biotechnological innovations with traditional fungal treatment systems, which may significantly improve feasibility and scalability.

 

The concluding section clearly summarizes the review's main points and highlights the importance of white-rot fungi in the biodegradation of synthetic dyes. However, this section could be improved:

The conclusion could more clearly summarize the key scientific findings of the review, such as the comparative effectiveness of the main ligninolytic enzymes or the most promising fungal species for dye degradation. Also, it would be useful to briefly highlight the most promising technological approaches, such as enzyme immobilization, the use of fungal consortia, or combined treatment systems combining biological and physicochemical methods.

R.: Thank you for this helpful comment. The conclusion section was revised to provide a clearer summary of the key scientific findings discussed throughout the review. In particular, we now highlight the comparative roles of the main ligninolytic enzymes involved in dye degradation and identify fungal species that have shown promising potential for this application.

 

Lines 692-728: Synthetic dyes are widely used worldwide due to their advantages, such as variability and durability. However, when improperly disposed of or inadequately treated, these compounds are released into ecosystems, causing significant harm to living organisms. This scenario highlights the urgent need for effective, low cost treatment methods that minimize environmental impacts while promoting the removal of xenobiotic compounds and water decolorization. In this context, bioremediation has emerged as a promising strategy, particularly through the use of white-rot fungi. The application of these organisms, either alone or in combination with physicochemical processes, represents an environmentally sustainable alternative for the treatment of textile effluents, characterized by low operational cost and high bioavailability. Evidence indicates that the oxidative enzymes produced by white-rot fungi, particularly lignin peroxidase, manganese peroxidase, versatile peroxidase, dye-decolorizing peroxidase, and laccase, exhibit remarkable efficiency in the degradation of dyes, including azo, anthraquinone and reactive, highlighting the central role of the ligninolytic enzymatic system in the oxidative breakdown of structurally complex chromophoric groups.

Despite limitations related to enzyme induction and secretion control, the fungal enzymatic system demonstrates a strong ability to degrade structurally complex pollutants, contributing to the recovery of environments impacted by textile industry discharges. Among the technological strategies discussed, the use of immobilized enzymes, fungal consortia, and hybrid treatment systems combining biological and physicochemical methods appears particularly promising for improving treatment performance. Nevertheless, additional studies under real scale conditions are essential to validate the industrial applicability of these biotechnological strategies. Greater attention should also be directed toward the development of fungal consortia, particularly those involving compatible WRF species capable of establishing synergistic rather than competitive interactions. Such relationships may stimulate enzymatic secretion and improve pollutant degradation performance. Moreover, the use of low cost substrates and agro industrial extracts as enzymatic inducers represents a promising pathway to increase economic feasibility while maintaining high treatment efficiency. Advancing research in these areas, together with improved understanding of enzymatic mechanisms and operational parameters, may help bridge the gap between laboratory findings and large-scale implementation. Despite these challenges, bioremediation using white-rot fungi represents a promising and sustainable strategy for the treatment of textile effluents and the mitigation of environmental pollution.

 

Overall assessment and recommendation

Overall, the article is a relevant, scientifically sound, and practically relevant review. It is well-structured, analytical, and covers contemporary literature. Recommended for publication after minor editorial revisions, taking into account the comments made.

R.: We sincerely thank the reviewer for the positive evaluation of our manuscript and for the constructive comments provided. We greatly appreciate the recognition of the relevance, structure, and scientific quality of our review. All the suggestions were carefully considered, and the manuscript was revised accordingly to address the points raised.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors The manuscript brings together a broad body of work on dye removal using white-rot fungi and ligninolytic enzymes. It covers the expected themes—key enzymes, operating conditions, sorption, and reported applications—and the structure is mostly clear, so the paper is easy to read as an entry point to the topic. The main limitation is that it currently functions more as a catalogue of studies than as a critical review that helps readers judge what works, under which conditions, and how reliable the evidence is. For that reason, I recommend major revision focused on strengthening the interpretation and increasing practical relevance. Major points 1. In multiple sections, “removal” is discussed largely in terms of color loss. For many dyes, this can be driven by biosorption or incomplete transformation, and it may generate products that are as toxic as—or more toxic than—the parent compound (e.g., aromatic amines from azo dyes). The manuscript should therefore avoid using decolorization as a stand‑in for degradation/mineralization/detoxification. Where possible, emphasize studies that report additional evidence (COD/TOC reduction, identification of transformation products, and at least one toxicity endpoint), and be explicit when the only endpoint is color. 2. The enzyme subsection is informative but reads as separate descriptions of laccase, LiP, MnP, etc. That format does not help a reader predict performance for a given dye or wastewater. A more useful approach would be to organize part of the section around dye classes (azo, anthraquinone, triarylmethane, etc.) and summarize: what typically works, what commonly fails, and the usual reasons (pH and salinity constraints, chloride inhibition, metal effects, mediator requirements, and peroxide control for peroxidases). A short “rules of thumb” table would add real value. 3.The scale-up discussion stays high-level, and much of the evidence cited appears to come from model solutions. Textile effluents are usually saline, contain surfactants and auxiliaries, and systems are non-sterile—conditions under which performance often drops. The paper should discuss matrix effects (salts/surfactants/metals), operational robustness under non-sterile conditions, and feasible reactor/operation modes (batch vs continuous; immobilized biomass; immobilized enzyme with reuse). Even a concise comparison table contrasting “synthetic dye solution” vs “real wastewater” outcomes would make the message more credible. 4. Since biosorption is included, the limitations need to be stated clearly: desorption risk, handling/disposal of dye-loaded biomass, and whether regeneration is practical. When reporting “removal efficiency,” clarify whether the dominant mechanism is adsorption or transformation, and highlight what should be reported for fair comparison (desorption/regeneration, effect of salinity and typical co-solutes, multi-cycle performance). 5. Please define and consistently use the terms decolorization, degradation/transformation, mineralization, and detoxification. Also check the reference list carefully (years, volumes, pages, DOIs). These are small issues individually, but together they affect confidence in a review article.

Author Response

Response to reviewers’ comments:

 

Reviewer 2:

The manuscript brings together a broad body of work on dye removal using white-rot fungi and ligninolytic enzymes. It covers the expected themes—key enzymes, operating conditions, sorption, and reported applications—and the structure is mostly clear, so the paper is easy to read as an entry point to the topic. The main limitation is that it currently functions more as a catalogue of studies than as a critical review that helps readers judge what works, under which conditions, and how reliable the evidence is. For that reason, I recommend major revision focused on strengthening the interpretation and increasing practical relevance.

R.: We appreciate the reviewer’s careful evaluation and constructive remarks. The manuscript was revised to strengthen interpretative discussion and to improve the practical relevance of the review. We expanded several sections to provide a more critical analysis of the reported studies and to better highlight the conditions influencing the effectiveness of fungal dye removal systems.

 

Major points:

1. In multiple sections, “removal” is discussed largely in terms of color loss. For many dyes, this can be driven by biosorption or incomplete transformation, and it may generate products that are as toxic as—or more toxic than—the parent compound (e.g., aromatic amines from azo dyes). The manuscript should therefore avoid using decolorization as a stand‑in for degradation/mineralization/detoxification. Where possible, emphasize studies that report additional evidence (COD/TOC reduction, identification of transformation products, and at least one toxicity endpoint), and be explicit when the only endpoint is color.

R: Thank you for this insightful observation. We agree that decolorization alone does not necessarily indicate complete degradation, mineralization, or detoxification of synthetic dyes. In the revised manuscript, we have clarified the terminology and emphasized the distinction between decolorization and actual chemical transformation.

 

2. The enzyme subsection is informative but reads as separate descriptions of laccase, LiP, MnP, etc. That format does not help a reader predict performance for a given dye or wastewater. A more useful approach would be to organize part of the section around dye classes (azo, anthraquinone, triarylmethane, etc.) and summarize: what typically works, what commonly fails, and the usual reasons (pH and salinity constraints, chloride inhibition, metal effects, mediator requirements, and peroxide control for peroxidases). A short “rules of thumb” table would add real value.

R.: We thank the reviewer for this valuable suggestion. While we maintained the original structure, which was praised by reviewer 1, describing the main ligninolytic enzymes to preserve the mechanistic perspective of the section, we have added a paragraph linking enzyme performance to common dye classes and included a new summary table (Table 2) highlighting general trends, limitations, and operational considerations for different dye types. This addition aims to improve the practical interpretation of the enzymatic degradation mechanisms.

 

Lines 361-375: While the enzymes described above have been extensively studied individually, their effectiveness in dye removal often depends on the chemical structure of the target compound and on the physicochemical conditions of the wastewater. Therefore, from an application-oriented perspective, it is also useful to consider how different classes of dyes typically respond to fungal enzymatic systems. In general, azo dyes are frequently de-graded through reductive cleavage of the azo bond followed by oxidative transformations (Ngo and Tischler, 2022), whereas anthraquinone and triarylmethane dyes often require strong oxidative enzymes such as laccases or peroxidases (Mohanty and Kumar, 2025; Pinto et al., 2026). However, the efficiency of these processes can be significantly influenced by environmental factors including pH, salinity, chloride concentration, the presence of metal ions, mediator availability for laccases, and the control of H₂O₂ levels in peroxidase-based systems. For practical purposes, some general trends reported in the literature are summarized in Table 2.

 

Table 2. General trends in fungal enzymatic degradation of common dye classes.

Dye class	Common limitations	Operational considerations	References
Azo	Formation of aromatic amines; incomplete mineralization	Often requires sequential anaerobic–aerobic processes or mediator systems	Chaturvedi et al., 2022; Tang et al., 2024
Anthraquinone	High structural stability; slower degradation rates	Higher redox potential enzymes often required	Jamal et al., 2022; Ming et al., 2022
Triarylmethane	Sensitivity to pH changes	Optimal activity typically in acidic conditions	Schroeder et al., 2025; Ullah et al., 2025
Reactive	High salinity in textile wastewater may inhibit enzymatic activity	Salinity and chloride levels should be considered	Lin et al., 2023; Sofia et al., 2024
Several dye classes in real effluents	Presence of metals, salts, and auxiliary chemicals	Mediator addition and peroxide control may enhance efficiency	Kumar et al., 2024; Negi et al., 2025

 

3.The scale-up discussion stays high-level, and much of the evidence cited appears to come from model solutions. Textile effluents are usually saline, contain surfactants and auxiliaries, and systems are non-sterile—conditions under which performance often drops. The paper should discuss matrix effects (salts/surfactants/metals), operational robustness under non-sterile conditions, and feasible reactor/operation modes (batch vs continuous; immobilized biomass; immobilized enzyme with reuse). Even a concise comparison table contrasting “synthetic dye solution” vs “real wastewater” outcomes would make the message more credible.

R.: We thank the reviewer for this insightful and constructive comment. In response, we expanded the discussion on scale-up considerations to better reflect the challenges associated with treating real textile wastewater. Specifically, we included a paragraph addressing the physicochemical complexity of industrial effluents, such as the presence of salts, surfactants, auxiliary chemicals, and non-sterile conditions, which can significantly influence fungal performance compared with laboratory experiments using synthetic dye solutions. Additionally, a new comparative table (Table 4) was included summarizing selected studies that evaluate fungal treatment systems using either synthetic dye solutions or real textile effluents.

 

Lines 652-666: Most studies evaluating fungal treatment of dye-containing wastewater are performed using synthetic dye solutions, which do not fully reflect the physicochemical complexity of real textile effluents. Consequently, treatment efficiencies observed under laboratory conditions may differ when applied to industrial wastewater. To illustrate this aspect, Table 4 compares selected studies employing fungal biomass or ligninolytic enzymes for dye removal in either synthetic or real effluents. Although these studies demonstrate the potential of fungal-based systems for dye degradation, the predominance of experiments conducted with synthetic matrices highlights an important research gap. Future investigations should therefore prioritize the use of real textile wastewater or more complex simulated effluents to better assess the robustness and practical applicability of these treatment strategies.

 

Table 4. Comparison of selected studies on fungal treatment of textile dyes using synthetic and real effluents.

Study	Treatment system	Effluent type	Key reported outcomes
Amaral et al., 2004	WRF Trametes versicolor producing extracellular oxidative enzymes (e.g., laccase) in batch cultures with glucose supplementation	Synthetic textile dye mixture and real textile wastewater from a dyeing facility	Up to 97% decolorization was achieved for synthetic dye mixtures, while ~92% removal was obtained for diluted real textile wastewater. More concentrated effluent showed lower efficiency (~40%), indicating inhibitory effects from wastewater constituents.
Thampraphaphon et al., 2022	Immobilized WRF Trametes hirsuta PW17-41 producing MnP as the main ligninolytic enzyme	Textile dye wastewater containing mixed industrial dyes	The immobilized fungal system achieved ~95.4% decolorization within 48 h, associated with MnP activity of ~ 4942 U L-1. The immobilization matrix improved operational stability and allowed biomass to be reused for up to 12 treatment cycles while maintaining high dye removal efficiency.
Johnnie et al., 2023	Laccase enzyme isolated from Pleurotus ostreatus	Textile industrial effluent containing dyes (e.g., Turquoise VG, Black B, Yellow R, Methyl Red), heavy metals, and auxiliary chemicals	Enzymatic treatment promoted significant dye decolorization and reductions in key physicochemical parameters, including COD, biological oxygen demand (BOD), total dissolved solids (TDS), turbidity, and conductivity.
Al-Rajhi et al., 2024	Biomass of the white-rot fungus P. chrysosporium used as a biosorbent for dye removal	Aqueous solutions containing Reactive Red and Reactive Blue dyes	Dead biomass removed ~82% of RR-198 and ~87% of RB-19 through biosorption. Optimal conditions included pH 3, 50 °C, 0.6 g adsorbent dosage, and 30 min contact time.

 

4. Since biosorption is included, the limitations need to be stated clearly: desorption risk, handling/disposal of dye-loaded biomass, and whether regeneration is practical. When reporting “removal efficiency,” clarify whether the dominant mechanism is adsorption or transformation, and highlight what should be reported for fair comparison (desorption/regeneration, effect of salinity and typical co-solutes, multi-cycle performance).

R.: We thank the reviewer for this important suggestion. New paragraphs discussing the main limitations of biosorption, including dye desorption risk, handling and disposal of dye-loaded biomass, and biosorbent regeneration, has been added to Section 5. Additionally, we clarified the distinction between dye removal by adsorption and enzymatic transformation and highlighted key parameters that should be reported for fair comparison among studies (e.g., regeneration capacity, multi-cycle performance, and the influence of salinity and co-solutes).

 

Lines 516-538: Despite its advantages, biosorption also presents important limitations that must be considered when evaluating its effectiveness for dye removal. One of the main concerns is the potential desorption of previously adsorbed dyes, particularly under changing environmental conditions such as pH, ionic strength, or temperature fluctuations (Tripathi et al., 2023). This reversibility can lead to the secondary release of pollutants into the treated effluent, limiting the long-term stability of the process.

Another critical aspect is the management of dye-loaded biomass generated after biosorption. Once saturated with contaminants, fungal biomass may require regeneration or safe disposal to avoid environmental risks. Although several regeneration strategies have been proposed, including chemical desorption or solvent washing, the efficiency of these approaches varies depending on the dye structure and the physicochemical characteristics of the biosorbent (Kuppusamy et al., 2026).

Furthermore, when reporting dye removal performance, it is essential to distinguish whether the observed removal efficiency is primarily due to adsorption (biosorption) or to enzymatic transformation and biodegradation. Since biosorption does not chemically alter dye molecules, removal percentages based solely on decolorization may overestimate the actual detoxification of the effluent (Schallemberger et al., 2023).

For a more reliable comparison between studies, future research should also report additional parameters such as biosorbent regeneration capacity, multi-cycle adsorption performance, the influence of salinity and competing solutes typically present in textile wastewater, and the potential for dye desorption under varying environmental conditions. Including these factors is essential for accurately assessing the practical applicability of biosorption processes in real wastewater treatment systems.

 

5. Please define and consistently use the terms decolorization, degradation/transformation, mineralization, and detoxification. Also check the reference list carefully (years, volumes, pages, DOIs). These are small issues individually, but together they affect confidence in a review article.

R.: Thank you for this helpful recommendation. We have defined and consistently applied the terms decolorization, degradation/transformation, mineralization, and detoxification to avoid ambiguity. Additionally, we carefully reviewed the entire reference list to verify the accuracy of publication.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The review is devoted to an important topic: the removal of dyes by fungal cultures and their enzymes. In my opinion, some data is insufficiently presented. For example, data on dye bleaching by the laccase/mediator system is not presented, although there is considerable research on this topic.

p.3 line 118 “White rot fungi are a group of microorganisms, mostly belonging to the phylum Basidiomycota…”All known fungi that cause white rot belong to the phylum Basidiomycota.

Figure 2. The source of the photos should be indicated.

p.5 line 167 “…they are also responsible for the reduction of Mn2+ to Mn3+..” Oxidation, not reduction.

p5. line 171 “Both fungi possess a similar ligninolytic enzymatic system mainly composed of laccase and manganese peroxidases…” The ligninolytic system of both fungi consists of laccase, manganese peroxidase and versatile peroxidase. (doi: 10.1371/journal.pone.0052446 doi: 10.1080/10826068.2015.1084513)

p.6 line 181 “…these fungi also produce aryl alcohol oxidase (AAO)…” If AAO is mentioned as a hydrogen peroxide producer, it is necessary to consider pyranose oxidase, glucose oxidase and glyoxal oxidase, which produce hydrogen peroxide.

p.6 line 192 “The dye-decolorizing peroxidase (DyP) activity consists of the decolorization of dyes and phenolic substrates. “The enzyme catalyzes the oxidation of non-phenolic substrates. Example - https://doi.org/10.3390/biom11091391

p.8 line 243 “The literature also reports that isoenzymes of P. eryngii and B. adusta exhibit a broader substrate specificity, being capable of oxidizing non-phenolic compounds and, therefore, functionally resembling LiPs (Hofrichter, 2002; Mäkinen et al., 2018)”.  These fungi have a versatile peroxidase. Ref Mäkinen et al., 2018 is not about P. eryngii and B. adusta.

p.8 line 251 Ref. Muñoz et al., 1997 This article is not about versatile peroxidase.

p.9 line 319 “The biodegradation of synthetic dyes into less toxic and colorless metabolites through ligninolytic enzymes      ” Is there any evidence that bleaching of dyes by ligninolytic enzymes results in non-toxic products? What products are formed as a result of such interaction?

Figure 4. “…dye removal by fungal systems…” “Plant cell” on the figure.

p.12 line 398 Section 5. “Biosorption by Fungal Biomass” Are there any data in the literature on specific cell structures on which dyes are adsorbed?

Author Response

Response to reviewers’ comments:

 

Reviewer 3:

The review is devoted to an important topic: the removal of dyes by fungal cultures and their enzymes. In my opinion, some data is insufficiently presented. For example, data on dye bleaching by the laccase/mediator system is not presented, although there is considerable research on this topic.

R.: We thank the reviewer for the careful reading of our manuscript. The comments and suggestions provided were very valuable and have been carefully considered in the revision of the manuscript, contributing to improving its clarity and overall quality.

            We thank the reviewer for this important observation. A new paragraph describing the role of laccase–mediator systems (LMS) in enhancing dye oxidation and decolorization has been added to Section 3.2 (Laccase). This addition highlights the mechanism of mediators, commonly used compounds, and their relevance for the degradation of recalcitrant dyes.

 

Lines 332-345: An important strategy to expand the oxidative capacity of laccases is the use of laccase–mediator systems (LMS). In these systems, low-molecular-weight redox mediators act as electron shuttles between the enzyme and substrates that cannot be directly oxidized due to steric or redox potential limitations. After being oxidized by laccase, the mediator forms reactive radical intermediates capable of attacking complex dye molecules, significantly increasing the range of degradable compounds (Zhang et al., 2024). Several synthetic mediators such as 1-hydroxybenzotriazole (HBT), ABTS, and violuric acid have been widely investigated, as well as natural mediators derived from lignin degradation (Gu et al., 2021; Li et al., 2022; Malcı et al., 2023). Numerous studies have demonstrated that LMS can substantially enhance the decolorization of recalcitrant dyes, including azo, anthraquinone, and indigoid dyes (Zosenko et al., 2022; Hordieieva et al., 2023; Otero et al., 2025; Younus et al., 2026). However, mediator toxicity, cost, and stability remain important challenges that must be considered when applying these systems in large-scale wastewater treatment (Rajendran et al., 2025).

 

p.3 line 118 “White rot fungi are a group of microorganisms, mostly belonging to the phylum Basidiomycota…”All known fungi that cause white rot belong to the phylum Basidiomycota.

R.: Thank you for pointing out this inaccuracy. The sentence has been corrected to indicate that all known white-rot fungi belong to the phylum Basidiomycota.

 

Figure 2. The source of the photos should be indicated.

R.: Thank you for this important observation. The images presented in Figure 2 are illustrative representations generated using the program Gemini based on morphological characteristics of the fungal taxa described in the literature.

 

p.5 line 167 “…they are also responsible for the reduction of Mn²⁺ to Mn³⁺.” Oxidation, not reduction.

R.: Thank you for the comment. The sentence was corrected to indicate the oxidation of Mn²⁺ to Mn³⁺.

 

p5. line 171 “Both fungi possess a similar ligninolytic enzymatic system mainly composed of laccase and manganese peroxidases…” The ligninolytic system of both fungi consists of laccase, manganese peroxidase and versatile peroxidase. (doi: 10.1371/journal.pone.0052446 doi: 10.1080/10826068.2015.1084513)

R.: Thank you for this observation. The sentence was revised to indicate that the ligninolytic system includes laccase, MnP, and VP. The suggested references were also incorporated to support this information.

 

p.6 line 181 “…these fungi also produce aryl alcohol oxidase (AAO)…” If AAO is mentioned as a hydrogen peroxide producer, it is necessary to consider pyranose oxidase, glucose oxidase and glyoxal oxidase, which produce hydrogen peroxide.

R.: Thank you for this observation. The text was revised to acknowledge additional oxidases involved in hydrogen peroxide generation. Specifically, pyranose oxidase, glucose oxidase, and glyoxal oxidase (GLOX) were included as potential extracellular sources of hydrogen peroxide that can supply the peroxide required for the catalytic activity of ligninolytic peroxidases.

 

p.6 line 192 “The dye-decolorizing peroxidase (DyP) activity consists of the decolorization of dyes and phenolic substrates. “The enzyme catalyzes the oxidation of non-phenolic substrates. Example - https://doi.org/10.3390/biom11091391

R.: Thank you for your comment. The sentence has been revised to clarify that DyPs can oxidize both phenolic and non-phenolic substrates, as reported in the literature. The suggested reference was incorporated to support this statement.

 

p.8 line 243 “The literature also reports that isoenzymes of P. eryngii and B. adusta exhibit a broader substrate specificity, being capable of oxidizing non-phenolic compounds and, therefore, functionally resembling LiPs (Hofrichter, 2002; Mäkinen et al., 2018)”.  These fungi have a versatile peroxidase. Ref Mäkinen et al., 2018 is not about P. eryngii and B. adusta.

R.: Thank you for pointing out this inconsistency. The references were carefully reviewed and corrected to ensure that they appropriately support the statements made in the text.

 

p.8 line 251 Ref. Muñoz et al., 1997 This article is not about versatile peroxidase.

R.: Thank you for pointing this out. The reference was reviewed and corrected.

 

p.9 line 319 “The biodegradation of synthetic dyes into less toxic and colorless metabolites through ligninolytic enzymes”. Is there any evidence that bleaching of dyes by ligninolytic enzymes results in non-toxic products? What products are formed as a result of such interaction?

R.: We thank the reviewer for this important and insightful comment. We agree that dye decolorization does not necessarily imply detoxification. Accordingly, the statement in the manuscript has been revised to clarify that enzymatic treatment by ligninolytic enzymes may lead to the formation of colorless metabolites, but not necessarily non-toxic products.

In addition, we have expanded this section to include a discussion of the main transformation products formed during enzymatic dye degradation. Specifically, we now mention that azo dyes are typically cleaved into aromatic amines, which may exhibit higher toxicity than the parent compounds.

 

Figure 4. “…dye removal by fungal systems…” “Plant cell” on the figure.

R.: Thank you for bringing this to our attention. The figure was revised to correct this labeling error and ensure that all elements are properly identified.

 

p.12 line 398 Section 5. “Biosorption by Fungal Biomass” Are there any data in the literature on specific cell structures on which dyes are adsorbed?

R.: We thank the reviewer for this relevant comment. We agree that a more detailed description of the structural basis of biosorption improves the mechanistic understanding of the process. Accordingly, we have added a new paragraph in Section 5 describing the main fungal cell wall components involved in dye adsorption. Specifically, the revised manuscript now highlights the role of structural polymers such as chitin, chitosan, and β-glucans, as well as associated proteins and lipids, which provide functional groups (e.g., amino, hydroxyl, carboxyl, and phosphate groups) that act as binding sites for dye molecules. The different interaction mechanisms involved, including electrostatic interactions, hydrogen bonding, and π–π interactions, are also now briefly discussed.

 

Lines 468-478: In fungal biomass, dye adsorption is primarily associated with specific structural components of the cell wall. The fungal cell wall is a complex matrix mainly composed of chitin, chitosan, β-glucans, proteins, and lipids, which provide a variety of functional groups such as amino, hydroxyl, carboxyl, and phosphate groups. These functional groups act as binding sites for dye molecules through mechanisms including electrostatic interactions, hydrogen bonding, van der Waals forces, and π–π interactions. Chitin and chitosan, in particular, play a key role due to the presence of amino groups that can interact strongly with anionic dyes, while glucans and associated proteins contribute to the overall adsorption capacity and structural stability of the biomass. Therefore, the composition and physicochemical properties of the fungal cell wall are critical factors influencing biosorption efficiency.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all the issues. Accept.