Yeast-Derived Biomolecules as Green Nanoplatforms for Sustainable Lignocellulosic Biorefineries

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Sanchez Vera et al. summarize in this review the role of yeast-derived biomolecules as green nanoplatforms for sustainable lignocellulosic biorefineries. The authors have divided the manuscript into four areas: (i) the expanding portfolio of metallic and metal-oxide NPs synthesized through yeast biomolecules; (ii) molecular-level mechanisms of reduction, capping, and surface tailoring that dictate NP morphology, stability, and reactivity; (iii) synergistic roles in intensifying lignocellulosic processes—from enhanced hydrolysis to catalytic upgrading; and (iv) frontier applications spanning antimicrobial coatings, regenerative packaging, precision agriculture, and environmental remediation. They conclude that yeast–biomolecule–driven nanoplatforms are not merely sustainable alternatives but transformative solutions for next-generation lignocellulosic biorefineries. Tables complement the review's message, and the cited literature is relevant and recent.

General comments:

The manuscript is written in the form of an interesting, generally clear, and well-structured review. Chapters and text have been utilized to their full extent.

In general, a well-done study that should be of interest to Fermentation readers. The article is an adequate, engaging novel and suitable for publication without any revisions.

 

Further comments:

Lines 15-16, 77-78, 88-89, and 253-254: The Authors repeat a part of the sentences four times…biosurfactants, exopolysaccharides, enzymes, pigments, proteins, and organic acids…in the whole manuscript. The reviewer's opinion is that the authors should reduce the number of repetitions of that part of the sentence.

Author Response

Reviewer´s comment: Sanchez Vera et al. summarize in this review the role of yeast-derived biomolecules as green nanoplatforms for sustainable lignocellulosic biorefineries. The authors have divided the manuscript into four areas: (i) the expanding portfolio of metallic and metal-oxide NPs synthesized through yeast biomolecules; (ii) molecular-level mechanisms of reduction, capping, and surface tailoring that dictate NP morphology, stability, and reactivity; (iii) synergistic roles in intensifying lignocellulosic processes—from enhanced hydrolysis to catalytic upgrading; and (iv) frontier applications spanning antimicrobial coatings, regenerative packaging, precision agriculture, and environmental remediation. They conclude that yeast–biomolecule–driven nanoplatforms are not merely sustainable alternatives but transformative solutions for next-generation lignocellulosic biorefineries. Tables complement the review's message, and the cited literature is relevant and recent.

 

General comments:

 

The manuscript is written in the form of an interesting, generally clear, and well-structured review. Chapters and text have been utilized to their full extent.

 

In general, a well-done study that should be of interest to Fermentation readers. The article is an adequate, engaging novel and suitable for publication without any revisions.

 

Response:

We thank Reviewer 1 for the positive overall assessment of our manuscript and for the constructive suggestion regarding repetition.

Comment 1

Lines 15–16, 77–78, 88–89, and 253–254: The authors repeat a part of the sentences four times… “biosurfactants, exopolysaccharides, enzymes, pigments, proteins, and organic acids” … in the whole manuscript. The reviewer’s opinion is that the authors should reduce the number of repetitions of that part of the sentence.

Response:
We appreciate this observation and agree that the repeated listing could be streamlined. In the revised version, we have:

• Retained the full list of biomolecule classes only where it is strictly necessary for clarity (e.g., at the beginning of Sections 4 and 5, where the scope is defined).
• Elsewhere, we shortened the expression (e.g., “yeast-derived biosurfactants and polysaccharides”, “yeast extracellular polymers and glycolipids”, “yeast-derived surface-active and redox-active metabolites”) to avoid redundancy.

These changes reduce repetition while preserving precision about the biomolecular classes discussed throughout the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a solid and coherent review on the role of yeast-derived biomolecules as sustainable nanoplatforms for green nanotechnology applied to lignocellulosic biorefineries.
The work effectively integrates yeast biotechnology with green nanotechnology under the circular bioeconomy framework. However, the critical discussion is primarily narrative and lacks quantitative comparative analysis.

Suggestions for modification:

1. Add a concise methodological section describing databases, keywords, review period, and inclusion/exclusion criteria.
2. Include comparative tables (e.g., nanoparticle yield, size, redox efficiency, cost, or stability) to strengthen the analytical consistency.
3. Enrich Sections 5 and 6 with mechanistic models, energy schemes, or metabolic pathways that link reduction, nucleation, and stabilization processes.
4. There is redundancy between Tables 1 and 2 on pretreatments. Additionally, the table format makes interpretation overly cumbersome. It is recommended to revise both tables.
5. Some abbreviations are undefined at first mention (e.g., NP, EPS, MEL).

Author Response

Reviewer´s comment: The manuscript presents a solid and coherent review on the role of yeast-derived biomolecules as sustainable nanoplatforms for green nanotechnology applied to lignocellulosic biorefineries.

The work effectively integrates yeast biotechnology with green nanotechnology under the circular bioeconomy framework. However, the critical discussion is primarily narrative and lacks quantitative comparative analysis.

Response: We are grateful to Reviewer 2 for the positive evaluation and for the specific suggestions, which have helped us significantly strengthen the analytical and mechanistic components of the review.

Comment 1

Add a concise methodological section describing databases, keywords, review period, and inclusion/exclusion criteria.

Response:
As suggested, we have added a concise methodological description at the end of the Introduction (Section 1). This new paragraph specifies:

• The databases used (Scopus and Web of Science as primary sources, complemented by targeted searches in Google Scholar for specific recent terms).
• The main keywords and combinations (e.g., “yeast”, “biosurfactant”, “exopolysaccharide”, “mannoprotein”, “green synthesis”, “nanoparticles”, “lignocellulosic biorefinery”, “circular bioeconomy”).
• The primary review period (last five years), with older, foundational references included when they provide mechanistic or historical context.
• The inclusion/exclusion criteria, focusing on peer-reviewed articles and reviews that provide mechanistic insight, quantitative data or explicit links between yeast systems, nanoparticle synthesis and biorefinery concepts, while excluding papers without clear experimental support, patents, and non-peer-reviewed sources.

This addition clarifies the methodological basis of the review and addresses the request for a more transparent and systematic literature selection.

Comment 2

Include comparative tables (e.g., nanoparticle yield, size, redox efficiency, cost, or stability) to strengthen the analytical consistency.

Response:
We fully agree with the need for more quantitative comparisons. In the revised manuscript we have:

• Introduced a new Table 4 (“Comparative physicochemical and functional metrics of yeast-mediated and chemically synthesized nanoparticles”), which directly compares yeast-derived versus chemically produced nanoparticles (Ag, ZnO, Se) in terms of:
• Particle size and size distribution (TEM, XRD, DLS).
• Colloidal stability (zeta potential, SPR band shape, qualitative aggregation reports).
• Antimicrobial/antioxidant performance at defined concentrations.
• Expanded the surrounding discussion in Section 5 to explicitly interpret these differences, emphasizing how yeast-derived biomolecular coronas influence size, dispersion, interfacial reactivity and functional performance compared with NaBH₄- or sol–gel-based routes.

Together with the existing Table 5 (which compiles yeast species, biomolecules, mechanisms and functional outcomes), the new Table 4 strengthens the analytical consistency of the manuscript and provides a clearer basis for mechanistic and techno-functional comparisons.

Comment 3

Enrich Sections 5 and 6 with mechanistic models, energy schemes, or metabolic pathways that link reduction, nucleation, and stabilization processes.

Response:
We have substantially revised and enriched Sections 5 and 6:

• In Section 5 (“Green Nanotechnology and the Role of Yeast Biomolecules”) we now include a mechanistic and energetic description of yeast-mediated nanoparticle synthesis as a “biological energy scheme”. This scheme explicitly links:

• Central carbon metabolism (glycolysis and the pentose phosphate pathway) to the generation of NADH/NADPH and redox-active metabolites (e.g., glutathione, quinones).
• Overflow metabolism and stress responses to the accumulation of organic acids and polyols that simultaneously chelate and reduce metal ions.
• The subsequent formation of M⁰ nanoclusters and their stabilization by biosurfactants, exopolysaccharides and mannoproteins.

In Section 6, we have also substantially expanded the comparison between extracellular and intracellular synthesis routes (Section 6.4), explicitly discussing their implications for downstream processing, scalability, energy demand, biological impurities and toxicological assessment. This revised subsection now clarifies why extracellular routes are generally more compatible with current biomanufacturing and regulatory frameworks, whereas intracellular routes are better suited for specialized, high-value applications (e.g. whole-cell catalysts and biosorbents).

Moreover, Section 6 now integrates process engineering aspects more explicitly: the revised subsection on process parameters (Section 6.5) links pH, precursor concentration, agitation and oxygen transfer to nucleation control, colloidal stability and yeast physiology, and connects these variables to design-of-experiments strategies and reactor-scale translation, thereby strengthening the bridge between mechanistic understanding and scalable bioprocess design.

We believe these additions directly address the reviewer’s request for mechanistic models and energy/pathway-based explanations.

Comment 4

There is redundancy between Tables 1 and 2 on pretreatments. Additionally, the table format makes interpretation overly cumbersome. It is recommended to revise both tables.

Response:
We appreciate this comment and have reorganized and clarified both tables:

• Table 1 now focuses specifically on pretreatment strategies → inhibitors → microbial impact → mitigation approaches in integrated biorefineries. The caption and introductory text have been revised to reflect this scope, and the table layout has been simplified for easier reading.
• Table 2 has been redefined to emphasize categories of pretreatment (physical, chemical, physicochemical, biological), their primary structural effects on biomass, and how these effects connect to downstream nanotechnology and valorization routes.

In addition, we have removed overlapping content between the two tables and improved formatting (column alignment, consistent terminology, clearer headings). The surrounding text clearly indicates the distinct purpose of each table, reducing redundancy and improving interpretability.

Comment 5

Some abbreviations are undefined at first mention (e.g., NP, EPS, MEL).

Response:
We have carefully checked the full manuscript to ensure that all abbreviations are defined at first use. In the revised version:

• “Nanoparticles (NPs)”,
• “Exopolysaccharides (EPS)”,
• “Mannosylerythritol lipids (MELs)”

are explicitly introduced at their first occurrence. We have also checked other recurring abbreviations (e.g., LCA, GRAS, DLS, SPR) and ensured consistent first-mention definitions.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The topic is potentially very significant, but it is partially treated too broadly and insufficiently critically, which means the declared goal of the work is not entirely fulfilled. Although the manuscript promises to fill a "critical gap" in the literature, it currently lacks an adequate, detailed overview of existing research and a critical assessment of it.

It is necessary to carry out a serious and systematic review of the literature, with a clear comparison, a critical assessment of earlier research results, and the definition of limitations and open questions in the field.

There is a lack of a clear and scientifically based explanation of the process of nanoparticle synthesis using yeasts, as well as an adequate review of the literature that would support it mechanistically and experimentally. This part is crucial to the work and must be significantly improved.

Also, there is a discrepancy between the listing of Table 1 (lines 118–120) and the actual content of the displayed table. The above description suggests a table of biomass structural fractions and valorization pathways, while the table actually shows pretreatment effects, inhibitors, and microbial responses. The tables need to be better formatted.

Particular attention should be paid to Chapter 4, which requires significant improvement and expansion, supported by a thorough literature review. Of additional concern is that the cited literature (e.g., reference 38, line 201) does not match the content claimed in the text. It is necessary to check whether such omissions occur in other places as well, because they damage the credibility of the manuscript.

In the Key challenges section (lines 290+), it is not enough to list the challenges; they need to be explained in detail, argued, and linked to aspects of industrial application and current technological limitations.

Finally, the paper does not provide a clear enough comparison of intracellular and extracellular nanoparticle synthesis. It is necessary to specify which approach is more industrially suitable, the main technological barriers, and the scalability, separation, toxicology, and economic viability associated with each approach.

Comments on the Quality of English Language

It is necessary to check and consistently use appropriate scientific and technical terminology. The terms "secrete", "workhorse", "toolkit" (and others) are either not used correctly or are stylistically unsuitable for a scientific text; they should be specified or replaced with appropriate professional terms.

Author Response

Reviewer´s comment: The topic is potentially very significant, but it is partially treated too broadly and insufficiently critically, which means the declared goal of the work is not entirely fulfilled. Cells for biopigment production. This research work is of industrially important and is suitable for consideration for publication in the journal.

Response: We thank Reviewer 3 for the thorough and critical evaluation. The comments have been very helpful for deepening both the mechanistic and critical aspects of the review.

Comment 1

The topic is potentially very significant, but it is partially treated too broadly and insufficiently critically. It is necessary to carry out a serious and systematic review of the literature, with a clear comparison, a critical assessment of earlier research results, and the definition of limitations and open questions in the field.

Response:
We fully agree with the reviewer that a more systematic and critical treatment is needed. In the revised manuscript, we have made the following changes:

1. Systematic literature basis:

• At the end of the Introduction, we now include a concise methodological description of our literature search (databases, keywords, time window, inclusion/exclusion criteria), clarifying the systematic nature of our review.

1. Quantitative comparisons and detailed overview of existing research:

• We have introduced Table 4, which provides quantitative comparisons between yeast-mediated and chemically synthesized nanoparticles (size, ζ-potential, stability, antimicrobial/antioxidant performance), and expanded Table 5 summarizing yeast species, biomolecular mediators, mechanisms and functional outcomes.
• Throughout Sections 4–7, we now systematically relate the main mechanistic findings and application case studies, highlighting consistencies and discrepancies across the literature instead of only describing individual studies.

1. Critical assessment and open questions:

• We have added and significantly expanded Section 8 (“Challenges and Limitations”), which is now subdivided into:

• 8.1 Biological variability and limited quantitative control
• 8.2 Scale-up, process integration and techno-economic realism
• 8.3 Regulatory, safety and environmental questions
• 8.4 Methodological fragmentation and poor comparability

In each subsection we critically examine the current literature, emphasize missing data (e.g., mass balances, volumetric productivities, techno-economic analyses, long-term toxicity, negative results), and clearly identify limitations and open questions that must be addressed for industrial implementation.

Collectively, these changes transform the manuscript from a primarily narrative overview into a more systematic and critically oriented review, aligned with the reviewer’s expectations.

 

Comment 2

There is a lack of a clear and scientifically based explanation of the process of nanoparticle synthesis using yeasts, as well as an adequate review of the literature that would support it mechanistically and experimentally. This part is crucial to the work and must be significantly improved.

Response:
We appreciate this important comment and have substantially revised the manuscript to address it. In the new version, Sections 4–6 have been expanded and reorganized to provide a clearer, mechanistically grounded description of yeast-mediated nanoparticle synthesis. Specifically, we now:

• Section 4: Provide a detailed, literature-supported description of how biosurfactants, exopolysaccharides, mannoproteins, enzymes, pigments and organic acids participate in metal reduction, nucleation and capping, with separate subsections (4.1–4.4) dedicated to each biomolecular class and their mechanisms of action.
• Section 5: Introduce a mechanistic and energetic “biological energy scheme” linking central carbon metabolism (glycolysis, pentose phosphate pathway, overflow metabolism) to the generation of redox-active metabolites, organic acids and ligands that drive nanoparticle formation, and discuss how process conditions modulate these pathways.

We have also added Table 4, which summarizes physicochemical and functional metrics for yeast-mediated versus chemically synthesized nanoparticles, thereby providing quantitative experimental support for the mechanistic discussion. Together, these changes significantly strengthen the mechanistic and experimental basis of the review.

 

Comment 3

There is a discrepancy between the listing of Table 1 (lines 118–120) and the actual content of the displayed table. The description suggests a table of biomass structural fractions and valorization pathways, while the table actually shows pretreatment effects, inhibitors, and microbial responses. The tables need to be better formatted.

Response:
We thank the reviewer for pointing out this inconsistency. In the revised manuscript:

• The caption and introductory sentence of Table 1 have been modified to match its actual content. Table 1 now clearly summarizes pretreatment strategies for lignocellulosic biomass, the main inhibitors generated, their impact on microbial performance, and the corresponding mitigation approaches in integrated biorefineries.
• The surrounding text has been adjusted to reflect this focus, and the formatting of both Table 1 and Table 2 has been improved (harmonized columns, clearer headings, reduced redundancy), so that each table has a distinct and coherent scope, as explained in our response to Reviewer 2.

 

Comment 4

Particular attention should be paid to Chapter 4, which requires significant improvement and expansion, supported by a thorough literature review. Of additional concern is that the cited literature (e.g., reference 38, line 201) does not match the content claimed in the text. It is necessary to check whether such omissions occur in other places as well, because they damage the credibility of the manuscript.

Response:
We thank the reviewer for this critical observation. In the revised manuscript, Chapter 4 has been substantially expanded and reorganized into Sections 4.1–4.5, which now provide a more detailed, mechanism-oriented discussion of biosurfactants, exopolysaccharides, mannoproteins/enzymes, and pigments/organic acids as mediators of yeast-assisted nanoparticle synthesis. Each subsection is supported by an updated and more comprehensive literature review, including both mechanistic studies and experimental reports.

In addition, we have carefully re-checked all citations in this chapter and in the subsequent sections to ensure that each reference accurately supports the statements in the text. The specific mismatch previously noted by the reviewer for the former reference 38 has been corrected, and similar inconsistencies were removed wherever identified. We believe that these changes significantly improve both the scientific robustness and the overall credibility of the manuscript.

 

Comment 5

In the Key challenges section (lines 290+), it is not enough to list the challenges; they need to be explained in detail, argued, and linked to aspects of industrial application and current technological limitations.

Response:
We fully agree that the challenges section must go beyond a simple listing. In the revised manuscript, the previous brief discussion has been expanded into a dedicated Section 8 (“Challenges and Limitations”), with four subsections:

• 8.1 Biological variability and limited quantitative control – where we explain how changes in yeast strain, medium composition, stress conditions and inoculum preparation affect biomolecular profiles and NP properties; we also discuss the implications for reproducibility and regulatory compliance.
• 8.2 Scale-up, process integration and techno-economic realism – where we connect current lab-scale practices (small batches, simplified media, detoxified hydrolysates) to industrial realities (high solids, foaming, viscosity, inhibitor variability), and highlight the scarcity of mass balances, volumetric productivities and techno-economic analyses. We explicitly relate these gaps to industrial feasibility, drawing on recent TEA/LCA studies for biogenic nanomaterials.
• 8.3 Regulatory, safety and environmental questions – where we discuss the complexity introduced by biomolecular coronas, the lack of long-term environmental fate and toxicity data, and the need for safe-and-sustainable-by-design approaches and life-cycle assessment, particularly for applications in food, agriculture and environmental remediation.
• 8.4 Methodological fragmentation and poor comparability – where we critically examine heterogeneous methodologies (medium, inoculum, characterization tools), the scarcity of negative results, and the absence of standardized reporting. We link this to ongoing initiatives defining minimum information standards for nanomaterials and bio–nano systems, and propose similar guidelines for yeast-mediated nanoparticle synthesis.

These subsections provide the requested detailed, argument-based discussion and explicitly connect the identified challenges to industrial application and current technological limitations.

 

Comment 6 – English language and terminology

It is necessary to check and consistently use appropriate scientific and technical terminology. The terms "secrete", "workhorse", "toolkit" (and others) are either not used correctly or are stylistically unsuitable for a scientific text; they should be specified or replaced with appropriate professional terms.

Response:
We appreciate this stylistic and terminological advice. In the revised manuscript, we have carefully reviewed the language and made targeted changes to ensure a consistently professional scientific tone. In particular:

• Expressions such as “workhorses” have been replaced by more technical phrases such as “core fermentative platforms” or “central fermentation hosts”.
• The term “toolkit” has been replaced by formulations such as “sets of molecular mediators”, “molecular repertoires” or “biomolecular sets”, depending on context.
• The verb “secrete” has been used more cautiously and, where appropriate, replaced or complemented by formulations such as “produce and release into the extracellular environment”, “release to the culture medium” or “accumulate in the extracellular matrix”, to accurately reflect the underlying processes.

We also carried out a broader revision of the manuscript to remove colloquial turns of phrase and harmonize the technical terminology across sections.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript reviewed the yeast-derived biomolecules as green nanoplatforms for sustainable lignocellulosic biorefineries. I thought that this study was informative and benefited readers doing or starting this type of research. In my opinion, this manuscript was acceptable after the following issue was addressed.

Please provide some discussion on the difference of the obtained nanoparticles using yeast-derived biomolecules and traditional chemicals? Besides being sustainable, was there any other advantage of yeast-derived biomolecules for the synthesis of the corresponding nanomaterials?

Author Response

Reviewer 4: Comments

This manuscript reviewed the yeast-derived biomolecules as green nanoplatforms for sustainable lignocellulosic biorefineries. I thought that this study was informative and benefited readers doing or starting this type of research. In my opinion, this manuscript was acceptable after the following issue was addressed.

Response: We thank Reviewer 4 for the positive evaluation and for the specific question regarding the comparative advantages of yeast-derived biomolecules in nanoparticle synthesis.

Comment 1

Please provide some discussion on the difference of the obtained nanoparticles using yeast-derived biomolecules and traditional chemicals? Besides being sustainable, was there any other advantage of yeast-derived biomolecules for the synthesis of the corresponding nanomaterials?

Response:
We appreciate this important question and have addressed it explicitly in the revised manuscript, primarily in Section 5 and around Table 4.

1. Comparative discussion of nanoparticle properties:
   After Table 4, we now provide a dedicated paragraph that contrasts nanoparticles obtained via yeast-derived biomolecules with those produced by conventional chemical reducers (e.g., NaBH₄, sol–gel routes). We highlight that:

• Yeast-mediated Ag, ZnO and Se nanoparticles typically fall within the 5–30 nm range, with narrower size distributions and stable surface plasmon resonance bands, and often exhibit more negative ζ-potentials, indicative of improved colloidal stability.
• Chemically synthesized AgNPs under comparable conditions tend to display larger hydrodynamic diameters, broader SPR bands and, in some reports, comparable or lower antimicrobial activity at equal or higher doses.

These trends are explicitly illustrated using literature data compiled in Table 4.

1. Advantages beyond sustainability:
   We have also added a new paragraph (just after this comparison) that directly addresses the reviewer’s question about advantages other than sustainability. In this paragraph, we emphasize that yeast-derived biomolecules:

• Enable in situ biofunctionalization, because mannoproteins, exopolysaccharides and glycolipid biosurfactants form biologically meaningful coronas that can impart antimicrobial, antibiofilm, antioxidant or catalytic functions without additional post-synthesis modification steps.
• Reduce the presence of residual strong synthetic reductants (e.g., NaBH₄, hydrazine), thereby simplifying toxicological assessment and potentially lowering risks for applications in food, agriculture and biomedicine.
• Often improve colloidal stability in complex environments (saline solutions, protein-rich media), as biosurfactant- and mannoprotein-based shells provide robust electrosteric barriers and help preserve surface functionality where conventional capping agents may fail.

We believe these additions clarify that yeast-derived biomolecules not only offer a more sustainable route to nanoparticle synthesis, but also expand the design space toward nanostructures with intrinsically bioactive, biocompatible and application-tailored surface chemistries.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

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