Review Reports - A Preliminary Study on Polyurethane Foam Ingestion by Tenebrio molitor Larvae and Potential Effects of Depolymerising Bacterial Supplementation

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Dear Authors,

despite your changes in the writing and the useful preliminary results regarding the poliuretane, the manuscript is still not acceptable for being published as the study is missing of accurate information regarding the microbial provision and the ratios of ingested feed.

Author Response

Comment 1.1. Dear Authors,

Author’s response: We sincerely thank the reviewer for the evaluation of our manuscript and for acknowledging the potential value of our preliminary findings. We understand the concerns regarding the information provided on microbial supplementation and polyurethane intake. In this revised version, we have further clarified the bacterial dose administered, the basis used to express bacterial concentrations, and the methodology used to estimate polyurethane intake. We have also expanded the discussion of the limitations associated with these measurements. While we acknowledge the exploratory nature of the study, we believe that the additional methodological details and the more explicit discussion of its limitations improve the transparency and interpretation of the results.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Please find my detailed comments and suggestions in the attached review report. I hope they will be helpful in further improving the manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English could be improved.

Author Response

Reviewer 2

Comment 1.2. Dear Authors, the manuscript addresses a timely and environmentally relevant topic by examining the ingestion of polyurethane foam by Tenebrio molitor larvae and exploring whether supplementation with selected depolymerising bacteria may influence larval performance or polyurethane consumption. The study shows potentially useful preliminary observations and is generally clearly structured. I particularly appreciate the authors’ cautious distinction between polyurethane ingestion, physical fragmentation, and true biodegradation, since the latter cannot be demonstrated without direct chemical characterisation of the polymer and its transformation products. The authors now appropriately acknowledge the exploratory nature and principal limitations of the work. Nevertheless, several methodological and interpretative aspects would benefit from further explanation to ensure that the experimental design is fully transparent and that all conclusions remain closely aligned with the evidence presented.

Author’s response: We thank the reviewer for the positive and constructive assessment of our work. We have carefully considered all comments and suggestions, which helped us improve the clarity of the methodology and the interpretation of the results. Detailed responses to each specific comment are provided below.

Comment 2.2. Lines 122–144: The bacterial culture in the PUBP treatment was administered as pieces of nutrient agar. Clarify whether the PUB treatment received an equivalent amount of sterile, uninoculated agar. If not, agar constitutes an additional variable between the PUB and PUBP treatments and may have provided nutrients to the larvae. Therefore, any numerical differences between these groups cannot be attributed solely to bacterial supplementation. This limitation should be explicitly acknowledged in the Materials and Methods, Discussion, and Conclusions.

Author’s response: We thank the reviewer for this important observation. The PUB treatment did not receive an equivalent amount of sterile agar, and therefore we agree that agar constituted an additional variable between the PUB and PUBP treatments. However, the amount of agar supplied was very small, resulting in a total supply of approximately 19.2 mg agar per tray and week (around 0.32 mg per larva and week). Agar is generally considered a stable, non-toxic gelling agent with very low nutritional value and has been recommended as a moisture source in mealworm feeding trials (Deruytter et al., 2025). Therefore, we considered its nutritional contribution unlikely to substantially affect larval performance. Nevertheless, we agree that this represents a potential confounding factor. Accordingly, this limitation has now been explicitly acknowledged in the Materials and Methods (L 139-142) and Discussion (L 329-333) sections.

Comment 2.3. Lines 122–144: The bacterial concentrations are reported as CFU/g, but the basis of this expression is unclear. Specify whether these values refer to grams of agar, bacterial preparation, total substrate, or another basis. If possible, also report the approximate viable bacterial dose supplied per tray and per application.

Author’s response: Bacterial concentration was expressed as colony-forming units (CFU) per gram of agar, which was the support matrix used for bacterial administration. This has now been explicitly stated in the Materials and Methods section. In addition, the amount of inoculated agar supplied per tray and application has been reported, allowing readers to estimate the approximate bacterial dose administered throughout the experiment.

Comment 2.4. Lines 137–144: Because bacterial viability and metabolic activity were not determined at each administration, moderate the phrase “ensuring a consistent source of bacterial biomass.”

Author’s response: We appreciate this suggestion. Since bacterial viability and metabolic activity were not assessed after each administration, the original wording could overstate the level of control achieved. The sentence has therefore been revised to indicate that the periodic additions provided a regular source of bacterial inoculum throughout the experimental period.

Comment 2.5. Lines 153–164: Clarify how polyurethane intake was calculated and how polyurethane fragments recovered in the frass, attached to the trays or larvae, or lost during handling were accounted for. Because polyurethane particles were detected in the frass, the term “intake” should be clearly distinguished from polyurethane disappearance or unrecovered polyurethane mass.

Author’s response: Polyurethane intake was estimated as the difference between the amount initially supplied and the residual material recovered at the end of the experimental period. No visible polyurethane dust or loose fragments were observed in the trays outside the frass, suggesting that material losses during handling were negligible. Polyurethane particles recovered in the frass were considered as ingested material, as they had necessarily passed through the digestive tract. To avoid ambiguity, the manuscript has been revised to clarify that polyurethane intake represents an estimate based on polyurethane disappearance from the substrate (L 153-157) and should not be interpreted as evidence of polyurethane biodegradation or complete utilisation by the larvae (i.e., L 31, 36, 104, 162, 193, 210, in the R1 version).

Comment 2.6. Lines 95–100, 144–146, 269–283 and 369–374: Fresh carrot was provided to all treatments as a water source, but it also supplied organic matter and nutrients. Therefore, the expressions “polyurethane alone” and “in the absence of other nutritional substrates” are not entirely precise. Please consider using “in the absence of a conventional dry feed substrate” or a similar formulation and apply it consistently throughout the manuscript.

Author’s response: We agree with the reviewer. Although carrot was supplied primarily as a source of moisture, it also provided small amounts of organic matter and nutrients. Therefore, expressions such as “polyurethane alone” or “in the absence of other nutritional substrates” could be misleading. These expressions have been replaced throughout the manuscript by more precise wording, such as “as the only dry substrate” or “in the absence of a conventional dry feed substrate”.

Comment 2.7. Lines 103–109 and 167–174: Larval mass and mortality were recorded weekly, whereas the statistical analysis is described only in general terms. Please explain whether only the final values were analysed or whether the weekly observations were also included. If longitudinal measurements were analysed, the model should account for time and the repeated-measures structure, with the tray treated as the experimental unit.

Author’s response: We thank the reviewer for this comment. Weekly measurements of larval weight and mortality were analysed as repeated measures using the MIXED procedure of SAS, with sampling date considered as the repeated factor and tray as the experimental unit. A detailed description of the statistical approach and covariance structure selection has now been incorporated into the Statistical Analysis subsection (L 173-179).

Comment 2.8. Lines 55–81, 247–250, 278–283 and 326–344: Although the authors appropriately acknowledge that chemical biodegradation was not demonstrated, some wording still appears to imply polyurethane degradation, digestion, or utilisation. Please review these statements and clearly distinguish established findings from proposed mechanisms or hypotheses.

Author’s response: We thank the reviewer for this observation. The manuscript has been carefully revised to ensure a clear distinction between polyurethane ingestion, physical fragmentation, polyurethane disappearance and experimentally demonstrated biodegradation. Statements that could imply chemical degradation, digestion or utilisation have been reworded to better reflect the evidence available and to avoid interpretations that extend beyond the results of the present study (e.g., L 282-284, 327-329, 380-384, of the R1 version).

Comment 2.9. Lines 95–98 and 269–270: Please supply additional information on the polyurethane foam, including its manufacturer or source, product type, density and, if available, chemical composition and additives. Without such characterisation, the statement that the foam contained no potentially nutritional components may be too definitive and should be moderated.

Author’s response: We thank the reviewer for this suggestion. Additional information regarding the polyurethane foam has now been incorporated in the Materials and Methods section (L 92-94). The material was a commercial semi-rigid polyurethane foam supplied to AITIIP by an industrial partner, presenting an apparent density of 40 kg/m³ and a closed-cell structure. Unfortunately, detailed information regarding its precise chemical composition and additives was not available. Therefore, statements assuming that the foam contained no potentially nutritional compounds have been moderated accordingly.

Comment 2.10. Lines 346–368 and 374–381: The environmental implications of physical fragmentation deserve detailed consideration. In the absence of demonstrated depolymerisation or mineralisation, fragmentation may generate smaller polyurethane particles rather than provide an effective waste-management solution. The authors acknowledge this concern in the Conclusions, but they should incorporate it more clearly into the Discussion.

Author’s response: We agree with the reviewer that the environmental implications of polyurethane fragmentation deserve further discussion. Although this aspect was briefly acknowledged in the previous version of the manuscript, it has now been discussed in greater detail. As the present study did not quantify the size distribution or abundance of particles excreted in the frass, the possibility that ingestion results mainly in fragmentation rather than complete biodegradation represents an important consideration. Accordingly, the Discussion section has been expanded to more explicitly address the potential generation of smaller polyurethane particles, the associated environmental concerns, and the need for future studies combining biological assays with chemical characterization of degradation products.

Comment 2.11. Overall, this interesting preliminary study may provide a useful basis for further research on insect-assisted polyurethane processing. The manuscript is generally balanced, and the authors appropriately avoid treating polyurethane disappearance or fragmentation as definitive evidence of biodegradation. However, the authors need to clarify the experimental controls, particularly the administration of the bacterial culture on nutrient agar, the bacterial dose, the calculation of polyurethane intake, and the statistical treatment of the weekly measurements. These issues do not necessarily diminish the study's exploratory value, but the authors should address them transparently and consistently throughout the manuscript. Subject to satisfactory clarification and appropriate revision of the relevant statements, I believe that the manuscript could be suitable for publication.

Author’s response: We sincerely thank the reviewer for the positive overall evaluation of our work and for recognising its potential value as a preliminary contribution to this research field. We believe that the modifications made in the text regarding bacterial administration, bacterial dose, polyurethane intake calculations and statistical analyses have substantially strengthened the manuscript and we greatly appreciate the reviewer’s constructive feedback.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a highly relevant and timely topic concerning the potential role of the mealworm Tenebrio molitor in the biodegradation of polyurethane foam, along with the possible enhancement of degradation efficiency through supplementation with depolymerizing probiotic bacteria. The study explores an innovative strategy for polyurethane waste management and offers promising perspectives for sustainable bioconversion of recalcitrant plastic materials. The topic is significant and aligns well with current interests in biological approaches to plastic degradation.

However, in its present form, the study appears to be at a preliminary stage, and the evidence provided is not yet sufficient to support the conclusions regarding polyurethane biodegradation.

The authors conducted two feeding experiments evaluating larval growth performance, substrate intake, and frass production under different dietary treatments. While these experiments provide useful preliminary insights into larval survival and feeding behavior in the presence of polyurethane, the evidence for actual polyurethane biodegradation remains inadequate. At present, the manuscript provides only microscopic images of polyurethane particles excreted in the frass of larvae fed polyurethane-containing diets. These observations demonstrate ingestion and physical fragmentation of polyurethane foam but do not confirm chemical degradation or depolymerization.

To substantiate the claim of biodegradation, it is essential that the authors provide FTIR analysis comparing the spectra of the original polyurethane foam with that of the frass residues. FTIR analysis would allow detection of alterations in characteristic polyurethane functional groups, thereby providing primary evidence of chemical modifications occurring during passage through the larval digestive system. Without such analysis, it is not possible to distinguish between simple mechanical fragmentation (by chewing) and actual polymer degradation. Furthermore, GPC and GC–MS analysis of degradation products would substantially strengthen the manuscript.

Another concern relates to the bacterial identification procedure described in lines 118–121, where the authors state that the bacterial strains were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). This statement requires clarification. Was MALDI-TOF MS employed for bacterial identification based on protein mass spectral fingerprinting, particularly ribosomal protein profiles compared against reference database entries? The authors should clarify whether the bacterial strains were identified through this protein fingerprint-based approach and provide sufficient methodological details regarding the database or identification platform used. Without this clarification, the description of bacterial identification remains ambiguous. Additionally, Figure 3a requires inclusion of a scale bar, as the current image lacks dimensional reference, making it difficult to interpret the size of the polyurethane particles shown.

Author Response

COMMENT: This manuscript addresses a highly relevant and timely topic concerning the potential role of the mealworm Tenebrio molitor in the biodegradation of polyurethane foam, along with the possible enhancement of degradation efficiency through supplementation with depolymerizing probiotic bacteria. The study explores an innovative strategy for polyurethane waste management and offers promising perspectives for sustainable bioconversion of recalcitrant plastic materials. The topic is significant and aligns well with current interests in biological approaches to plastic degradation.

However, in its present form, the study appears to be at a preliminary stage, and the evidence provided is not yet sufficient to support the conclusions regarding polyurethane biodegradation. The authors conducted two feeding experiments evaluating larval growth performance, substrate intake, and frass production under different dietary treatments. While these experiments provide useful preliminary insights into larval survival and feeding behavior in the presence of polyurethane, the evidence for actual polyurethane biodegradation remains inadequate. At present, the manuscript provides only microscopic images of polyurethane particles excreted in the frass of larvae fed polyurethane-containing diets. These observations demonstrate ingestion and physical fragmentation of polyurethane foam but do not confirm chemical degradation or depolymerization.

RESPONSE: We thank the reviewer for his constructive assessment of our manuscript, as well as for recognising the relevance and novelty of the proposed approach. We agree with the reviewer that the present study represents an initial stage in the evaluation of polyurethane utilisation by Tenebrio molitor larvae, and that the evidence provided does not allow for definitive confirmation of polyurethane biodegradation. The primary objective of this work was to perform a preliminary exploratory assessment to determine whether larvae are capable of ingesting polyurethane foam under controlled conditions, and whether supplementation with depolymerising bacteria could elicit a measurable biological response. As correctly indicated, the microscopic observations of polyurethane particles in the frass demonstrate ingestion and physical fragmentation, but do not constitute direct evidence of chemical degradation or depolymerisation. We fully acknowledge this limitation, and in this way we have revised the manuscript to clarify the exploratory nature of the study avoiding overinterpretation of the results. Specifically:

The title, simple summary, abstract and conclusions sections have been moderated to clearly state that polyurethane biodegradation could not be confirmed under the conditions of this study.
The terminology throughout the manuscript has been adjusted to distinguish between ingestion, fragmentation, and biodegradation.
Additional text has been included in the discussion to highlight the need for further analyses (e.g., chemical characterisation of degradation products) to confirm biodegradation mechanisms.

We believe that, fitted to this revised framework, the study provides useful preliminary evidence that may support future research aimed at improving the efficiency of biological polyurethane degradation strategies.

COMMENT: To substantiate the claim of biodegradation, it is essential that the authors provide FTIR analysis comparing the spectra of the original polyurethane foam with that of the frass residues. FTIR analysis would allow detection of alterations in characteristic polyurethane functional groups, thereby providing primary evidence of chemical modifications occurring during passage through the larval digestive system. Without such analysis, it is not possible to distinguish between simple mechanical fragmentation (by chewing) and actual polymer degradation. Furthermore, GPC and GC–MS analysis of degradation products would substantially strengthen the manuscript.

RESPONSE: We thank the reviewer for this technically founded suggestion. We fully agree that analytical techniques such as FTIR, GPC, and GC–MS would provide essential information to assess chemical modifications in polyurethane and to distinguish between mechanical fragmentation and actual polymer degradation. As correctly indicated, FTIR analysis comparing the spectra of the original polyurethane material and the frass residues would allow detection of potential alterations in characteristic functional groups, while GPC and GC–MS analyses would further contribute to identifying changes in polymer molecular weight distribution and the presence of degradation products.

However, the present study was conceived as an exploratory step aimed at evaluating 1) the ability of Tenebrio molitor larvae to ingest polyurethane foam under controlled conditions, and 2) whether supplementation with a culture of depolymerising bacteria could induce a measurable biological response. Within this framework, the experimental design focused on larval performance, intake, and frass production as primary indicators. We agree that the absence of chemical characterisation techniques may represent a limitation of the current study, as it is now commented throughout text. The discussion section has been expanded to highlight the need for FTIR, GPC, GC–MS, or similar analytical approaches in future studies to confirm polyurethane biodegradation and elucidate the underlying mechanisms. Accordingly, the conclusions have been revised to avoid overinterpretation, clearly stating that polyurethane biodegradation could not be confirmed under the conditions of this study. We believe that, despite this limitation, the results provide useful preliminary evidence that may serve as a basis for more detailed mechanistic investigations.

COMMENT: Another concern relates to the bacterial identification procedure described in lines 118–121, where the authors state that the bacterial strains were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). This statement requires clarification. Was MALDI-TOF MS employed for bacterial identification based on protein mass spectral fingerprinting, particularly ribosomal protein profiles compared against reference database entries? The authors should clarify whether the bacterial strains were identified through this protein fingerprint-based approach and provide sufficient methodological details regarding the database or identification platform used. Without this clarification, the description of bacterial identification remains ambiguous.

RESPONSE: We agree with the need to clarify the bacterial identification procedure. The bacterial strains used in this study were provided by CETEM (Murcia, Spain) and were previously isolated and identified following the methodology described in Muñoz-Martí et al. (2025). As indicated in that study, identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based on protein mass spectral fingerprinting, primarily reflecting ribosomal protein profiles, which were compared against reference database entries. Briefly, bacterial isolates were analysed using a Microflex L20 system (Bruker Daltonics, Fremont, CA, USA) equipped with an N₂ laser, applying the ‘extended direct transfer’ method. Spectra were acquired in positive linear ion mode with an acceleration voltage of 20 kV. Each spectrum corresponded to the sum of 240 laser shots, within a mass range of 2000–20,000 Da, and three spectra per strain were obtained. Identification was performed using the MALDI Biotyper Realtime Classification method against the MBT 7854 and MBT 7311_RUO (Bruker Daltonics) databases, as well as the MALDI-TOF database of the Spanish Type Culture Collection (CECT) and the Drinking Water Library (DWL).

This information has now been clarified in the revised manuscript (L116-131 of the R1 version).

COMMENT: Additionally, Figure 3a requires inclusion of a scale bar, as the current image lacks dimensional reference, making it difficult to interpret the size of the polyurethane particles shown.

RESPONSE: We thank the reviewer for this comment. A scale bar has been added to Figure 3a to facilitate interpretation of particle size.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for providing this research study on the

Provide detailed information on the protocol for preparing the probiotic culture as their addition is the key point of the study. As the bacteria were not freshly preparated, how can you assume that their metabolic input was the same at the different time of provision ?

L136-137 F:G is FCR feed conversion ratio ?

The study does not provide information related to the microbial community of the insect during the rearing and the consumption of the different treatment, a key information for understanding the difference in larval growth amongst treatments.

Also larval composition in terms of macronutrients in needed for giving an overview on the occuring effect of the diet on the insects.

Despite is a well structured study, I suggest to consider to add and to perform other experiments for implementing the already present assumptions.

Kind regards

Author Response

COMMENT: Provide detailed information on the protocol for preparing the probiotic culture as their addition is the key point of the study. As the bacteria were not freshly prepared, how can you assume that their metabolic input was the same at the different time of provision ?

RESPONSE: We agree that the preparation and consistency of the probiotic culture are key aspects of the experimental design. In fact, in order to minimise variability, a single batch of agar plates containing the probiotic bacterial mixture was prepared, ensuring consistent initial bacterial concentration and culture conditions across all experimental replicates. These culture plates were stored at 4 °C until use, a common approach to slow down microbial metabolic activity and maintain cell viability over time. We acknowledge, however, that bacterial metabolic activity at the time of administration was not directly measured, and therefore some variation in physiological state over the storage period cannot be completely excluded. Nevertheless, the use of a single batch was intended to reduce variability associated with repeated culture preparation, such as differences in growth phase, medium composition or incubation conditions. Under this approach, all larvae in the PUBP group were exposed to a comparable starting material throughout the experimental period.

This aspect has been clarified in the revised manuscript (L131-134 of the R1 version).

COMMENT: L136-137 F:G is FCR feed conversion ratio ?

RESPONSE: The reviewer is correct, F:G and FCR could be considered as the same parameter. In this manuscript, we decided to use F:G to be in line with other studies within the field.

COMMENT: The study does not provide information related to the microbial community of the insect during the rearing and the consumption of the different treatment, a key information for understanding the difference in larval growth amongst treatments.

RESPONSE: We thank the reviewer for this insightful comment. We fully agree that characterisation of the microbial community within the larval gut would provide valuable information to better understand the differences observed in larval performance among treatments, particularly in relation to the potential establishment and activity of the supplemented bacterial strains. In the present study, the experimental approach was designed as an initial exploratory step focusing on larval growth performance, substrate intake, and frass production as primary indicators of response to polyurethane feeding and bacterial supplementation. Therefore, microbial community analysis was beyond the scope of the original objectives. Nevertheless, we acknowledge that the lack of microbiome characterisation represents a limitation of the current work. We have highlighted this aspect into the revised discussion (L 283-316 of the revised version), highlighting the need for future studies to evaluate the dynamics of the gut microbiota, including the potential colonisation and functional contribution of the supplemented bacteria. Such analyses would be essential to elucidate the mechanisms underlying the observed responses and to determine whether the tested bacterial strains can effectively establish within the digestive tract and contribute to polymer transformation.

COMMENT: Also larval composition in terms of macronutrients in needed for giving an overview on the occuring effect of the diet on the insects.

RESPONSE: We agree that the analysis of larval composition in terms of macronutrients could provide additional information into the effects of the different dietary treatments on nutrient utilisation and physiological status of the insects. However, our primary objective was to evaluate the capacity of Tenebrio molitor larvae to ingest polyurethane foam and to assess whether bacterial supplementation could induce a measurable response under conditions of limited nutrient availability. Within this context, the experimental design focused on survival, intake, and growth performance as primary indicators, but we did not consider the surviving bacteria as potential source of nutrients for animals or humans. It should also be noted that larval growth was minimal due to the restricted nutritional value of the experimental diets, particularly in treatments where polyurethane was the single substrate. Under these conditions, substantial changes in body composition were not expected. Nevertheless, we acknowledge that the inclusion of detailed compositional analyses in future studies will strengthen the overall characterisation of the biological response.

COMMENT: Despite is a well structured study, I suggest to consider to add and to perform other experiments for implementing the already present assumptions.

RESPONSE: We thank the reviewer for this positive assessment of the structure of the study and for the constructive suggestion. We agree that additional experiments would be valuable to further substantiate and expand the findings presented in this work. However, the present study was conceived as an initial exploratory step aimed at evaluating the potential of Tenebrio molitor larvae to ingest polyurethane foam and to assess the possible response to bacterial supplementation under controlled conditions and thus the objective was to generate preliminary evidence rather than to provide a comprehensive validation of the proposed strategy. We fully acknowledge that further experimental work, including more detailed mechanistic and analytical approaches, will be necessary to confirm and extend the current findings. This perspective has been incorporated into the revised manuscript, where the need for additional studies is clearly highlighted. We believe that, in its current form, the manuscript provides a useful basis for future research exploring biological approaches to polyurethane degradation.