Black Soldier Fly Can Safely Co-Convert Antibiotic Fermentation Residue and Potato Peel Waste into a Valuable Feed Resource

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript (insects-3974275, Black Soldier Fly Can Safely Co-convert Antibiotic Fermentation Residue and Potato-Peel Waste into a Valuable Feed Resource) describes the co-bioconversion of two under-utilized industrial side-streams—Nosiheptide fermentation residue (NFR) and potato-peel waste (PPW)—by black soldier fly larvae (BSFL). The authors optimize the NFR:PPW ratio (3:5 dw) and the larva-to-feed ratio (1.5:1) to achieve 40 % mass reduction, 8.6 % bioconversion, and 55–58 % Nosiheptide degradation. They further characterize the microbial succession in larval guts and substrate, quantify residual antibiotic in de-fatted larval meal, and evaluate amino-acid and fatty-acid profiles. The work is timely and fits the scope of Insects.

However, several issues—ranging from mechanistic depth and safety assessment to data presentation and language—must be addressed before publication can be recommended.

Introduction

1) “the efficacy of BSFL in converting Nosiheptide fermentation residue… remain inadequately understood”. The gap is generic; the authors do not indicate why Nosiheptide (a thiopeptide) is different from tetracyclines or ciprofloxacin already studied with BSFL.

2) Only parent Nosiheptide concentration is monitored (Fig. 2). Essential to identify transformation products and verify loss of antimicrobial activity; otherwise “degradation” could be mere adsorption.

3) Residue Level vs Regulatory Threshold. Abstract claims “potential safety for animal feed” because residual Nosiheptide is 25 mg kg⁻¹. This point should be clearly stated.

4) Fig. 4D: 1.5:1 vs 2:1 bars carry different letters, but text states “incremental gain … no longer significant (p > 0.05)”? Verify post-hoc Tukey test; if p > 0.05, assign identical letters and add “ns” symbol.

5) Nosiheptide extraction: Add spike-recovery test.

6) Provide error values in text, not only in figures.

7) In discussion. Avoid repeating abstract numbers verbatim; instead explain why 55 % is a breakthrough for thiopeptides. Discuss energy/carbon footprint versus incineration (brief) to align with “dual-carbon” context mentioned in Introduction.

Author Response

Comment1: “the efficacy of BSFL in converting Nosiheptide fermentation residue… remain inadequately understood”. The gap is generic; the authors do not indicate why Nosiheptide (a thiopeptide) is different from tetracyclines or ciprofloxacin already studied with BSFL.

Response1: Thank you very much to the reviewers for their valuable comments. Currently, in animal husbandry and aquaculture, nosiheptide promotes nutrient absorption and weight gain in livestock and poultry by inhibiting the growth and reproduction of Gram‑positive bacteria. It has low toxicity in animals and causes little environmental pollution, and has been widely used in feed for poultry, livestock, and aquatic animals [1]. In 1998, nosiheptide was approved as a national Category III new veterinary drug in China. In 2017, the Ministry of Agriculture and Rural Affairs of China revised the "Directory of Varieties and Usage Specifications for Medicinal Feed Additives". However, with the extensive use of nosiheptide, its fermentation residues contain large amounts of the antibiotic, and degrading these antibiotics has become a challenge. To date, no reports have described particularly effective and low‑cost degradation methods. Corresponding explanations for the differences between tetracycline and nosiheptide have also been provided in the revised manuscript (see lines 90–94, 98–100, and 103–107). 

Comment 2: only parent Nosiheptide concentration is monitored (Fig. 2). Essential to identify transformation products and verify loss of antimicrobial activity; otherwise “degradation” could be mere adsorption.

Response2: We are grateful to the reviewer for taking the time to assess our work and provide valuable feedback. It is critical to evaluate the toxicity following antibiotic degradation. Accordingly, antibacterial assays were performed to analyze neomycin residues before and after BSFL-mediated degradation. Toxicity tests of antibiotics before and after degradation showed that the inhibitory effect on Bacillus was significantly reduced after degradation. The results are presented in Table 5 of the revised manuscript. The conclusion is presented in lines 368–371 of the revised manuscript. Our review of the relevant literature did not reveal any studies on the degradation products of Nosiheptide. Therefore, Q‑TOF mass spectrometry was used to identify the degradation products. However, no conclusive evidence regarding the types of degradation products has been obtained to date. This issue is also discussed in the revised manuscript and is presented in lines 633–645.

Comment 3: Residue Level vs Regulatory Threshold. Abstract claims “potential safety for animal feed” because residual Nosiheptide is 25 mg kg⁻¹. This point should be clearly stated.

Response3: The reviewer’s insightful suggestions have greatly helped improve the quality of our paper; we thank you sincerely. We subjected BSFL that had previously been exposed to nosiheptide to a prolonged intestinal emptying process to determine whether this treatment could reduce the nosiheptide content in their bodies. After 48 hours of gut emptying, no nosiheptide residues were detected in the insect powder, indicating that the residual nosiheptide had fallen below the detection limit. This conclusion has been added to lines 391–395 of the revised manuscript. In the preface section, supplementary information regarding the usage guidelines of nosiheptide is also provided. According to the latest Standard No. 246 issued by the Ministry of Agriculture and Rural Affairs of China, nosiheptide has been reclassified from a feed additive to a veterinary drug. Therefore, it can no longer be arbitrarily added to feed and may only be used as a veterinary therapeutic agent. Please find the related modification in lines 115-118.

Comment 4: Fig. 4D: 1.5:1 vs 2:1 bars carry different letters, but text states “incremental gain … no longer significant (p > 0.05)”? Verify post-hoc Tukey test; if p > 0.05, assign identical letters and add “ns” symbol.

Response 4: We thank the reviewer for pointing out these errors. This oversight has been corrected. The revision appears in line 351-352.

Comment 5: Nosiheptide extraction: Add spike-recovery test.

Response 5: We would like to express our sincere thanks to the reviewer for their critical and encouraging evaluation. The Add spike-recovery test was conducted with nosiheptide at different concentrations, and the experimental method was documented. The details are presented in lines 244–253 of the revised manuscript. The experimental results are presented in Table 4, and the corresponding description is provided in lines 296–297 of the revised manuscript.

Comment 6: Provide error values in text, not only in figures.

Response 6: We thank the reviewer for raising these important points, which have led to substantial improvements in our revision. The error values has been added to the data in the text, such as in lines 303-304 in the manuscript: “The lowest concentration was recorded at 126.68 ± 9.77 mg/kg in the R-8 group, while the highest concentration was observed at 150.76 ± 2.79 mg/kg in the R-2 group (Fig. 2A).

Comment 7: In discussion. Avoid repeating abstract numbers verbatim; instead explain why 55 % is a breakthrough for thiopeptides. Discuss energy/carbon footprint versus incineration (brief) to align with “dual-carbon” context mentioned in Introduction.

Response 7: We are very grateful for the valuable comments from the reviewers, which help improve the framework of our paper. We believe that this part is not the focus of this manuscript, so we have also reduced the content in this section, specifically in lines 62-63 in the revised manuscript. We also had a brief discussion on this issue, as detailed in lines 613-616 in the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Black soldier fly can safely co-convert antibiotic fermentation residue and potato peel waste into a valuable feed resource” investigates the co-conversion of antibiotic fermentation residue and potato peel waste by black soldier fly larvae (BSFL), aiming to determine the optimal ratio for Nosiheptide degradation and biomass conversion.

The topic is potentially valuable, as it explores an additional and timely application of BSFL in antibiotic degradation and organic waste valorization. However, the current version of the manuscript requires substantial improvement in both experimental design and scientific writing before it can be considered for publication.

In addition to several grammatical issues, the logical flow and clarity of the manuscript should be revised. The presentation of results and the framing of key arguments make it difficult for readers to follow the authors’ intended messages. I strongly recommend that the authors thoroughly re-examine the entire manuscript and consider using a professional English editing service.

Some major concerns were outlined below as examples:

1.  Inappropriate use of C/N ratio
   The C/N ratio was used as an indicator but the actual protein and carbohydrate (hydrocarbon) content may be more relevant indicators. Parameters such as protein/larvae or carbohydrate/larvae would provide more meaningful insights than the aggregated C/N ratio, which conceals important compositional details.
   
   
2. Missing methodology for crude protein estimation and gut clearance
   1. The manuscript does not describe the method used to estimate crude protein content. Without this information, it is impossible to evaluate the reliability of the reported values. Consequently, any conclusions drawn from these data are questionable and should be treated with caution.
   2. The procedure for gut clearance is not described in the Materials and Methods section. As a result, it is impossible to evaluate if the process was appropriate. Furthermore, the authors conclude that the residual Nosiheptide detected in larvae was due to “incomplete gut evacuation,” but no sufficient evidence is provided to support this explanation. Safety is one of the major elements of this study, a much more cautious and evidence-based conclusion is required.

While the topic is of interest, the manuscript in its current form suffers from major weaknesses in research design, data interpretation, and scientific writing. I recommend that the authors conduct a comprehensive revision to address these fundamental issues and ensure that all experimental methods, data justifications, and conclusions are scientifically sound.

Author Response

We are extremely grateful to the reviewers for their valuable suggestions for improving our article.

Comment1: Inappropriate use of C/N ratio
The C/N ratio was used as an indicator but the actual protein and carbohydrate (hydrocarbon) content may be more relevant indicators. Parameters such as protein/larvae or carbohydrate/larvae would provide more meaningful insights than the aggregated C/N ratio, which conceals important compositional details.

Response1: We deeply appreciate the reviewer’s expertise and the time they dedicated to providing such thorough feedback. Based on the analysis of protein, fat, and ash contents, the total protein and carbohydrate contents were calculated for each group. The relevant parameter conditions, such as the precise protein per larva, have been added to Table 1.

Comment 2: Missing methodology for crude protein estimation and gut clearance

1. 1. The manuscript does not describe the method used to estimate crude protein content. Without this information, it is impossible to evaluate the reliability of the reported values. Consequently, any conclusions drawn from these data are questionable and should be treated with caution.
   2. The procedure for gut clearance is not described in the Materials and Methods section. As a result, it is impossible to evaluate if the process was appropriate. Furthermore, the authors conclude that the residual Nosiheptide detected in larvae was due to “incomplete gut evacuation,” but no sufficient evidence is provided to support this explanation. Safety is one of the major elements of this study, a much more cautious and evidence-based conclusion is required.

Response 2: We appreciate the reviewer’s detailed and careful reading of our manuscript and their helpful remarks. We believe that the reviewers' suggestions are important.

1. 1. Specific additions have been made to the method for measuring protein content, and detailed in lines 137 to 143 of the revised manuscript.
   2. The intestinal cleaning method has been added in lines 194–199 of the revised manuscript.

In addition to the primary investigations, we have also carried out a series of supplementary experiments, including an antibacterial susceptibility test as well as a recovery test that involved the addition of Nosiheptide. The revised manuscript also describes the method for determining the fat content, specifically on lines 144–154. Furthermore, the entire manuscript has been carefully revised and thoroughly edited to ensure clarity and correctness in English expression.

Reviewer 3 Report

Comments and Suggestions for Authors

The work is certainly relevant and important. It concerns the acute problem of the widespread use of antibiotics in our lives, and especially the use of antibiotics in farm animal feed. This is due to the intensive production of products to provide food to the population. The need for antibiotics is caused by their large-scale production and the corresponding accumulation of industrial waste, the disposal of which is devoted to this work. The goals and objectives set by the authors are correct. And in principle, the work was done at a good professional level, combining the offer of a specialized feed substrate for larvae and its optimization, the analysis of the intestinal microbial community digesting the studied feed substrate, and the characteristics of the biomass of larvae in order to determine the possibility of their use in animal feed. It is concluded that the studied insect larvae are able to break down the antibiotic, and they can be used in feed. This correct conclusion follows from the presented experimental work. However, another conclusion follows from the data obtained. As written by the authors, "high-concentration antibiotic treatment induced significant microbial changes: the Bacteroidota phylum in the high-concentration gut group (HG8) increased significantly (45.13%), with Dysgonomonas becoming the dominant genus (38.20%), and its abundance was 2.9 times higher than that in the low concentration group (LG8), demonstrating its significant tolerance to the high-antibiotic environment." This means that when using the proposed feed for growing larvae, the number of antibiotic-resistant bacteria in their intestines increases significantly. So in frass (zoo compost) These antibiotic-resistant bacteria isolated from larvae with excrement will also be present.  Thus, antibiotic-resistant bacteria can enter the environment without special frass treatment. As a result, it is possible to obtain not a positive effect from reducing the antibiotic in industrial waste, but a negative effect from increasing the problem of obtaining antibiotic-resistant microorganisms. As well as the presence of antibiotic-resistant microorganisms in the larval biomass itself requires their sterilization after separation from the substrate. This issue is not discussed by the authors. Therefore, I consider it premature to conclude that "Collectively, these findings present a novel approach for the management of antibiotic fermentation residue waste through the utilization of BSFL." The paper should necessarily include a discussion or even additional experiments on the problem of obtaining Nosiheptide-resistant bacteria in the BSFL intestine and in the frass. It would be premature to publish an article without this.

Author Response

We would like to thank the reviewer for the time and effort spent on our manuscript. The comments are very helpful. Our point-by-point responses are as follows：

Comment 1: However, another conclusion follows from the data obtained. As written by the authors, "high-concentration antibiotic treatment induced significant microbial changes: the Bacteroidota phylum in the high-concentration gut group (HG8) increased significantly (45.13%), with Dysgonomonas becoming the dominant genus (38.20%), and its abundance was 2.9 times higher than that in the low concentration group (LG8), demonstrating its significant tolerance to the high-antibiotic environment." This means that when using the proposed feed for growing larvae, the number of antibiotic-resistant bacteria in their intestines increases significantly. So in frass (zoo compost) These antibiotic-resistant bacteria isolated from larvae with excrement will also be present.  Thus, antibiotic-resistant bacteria can enter the environment without special frass treatment. As a result, it is possible to obtain not a positive effect from reducing the antibiotic in industrial waste, but a negative effect from increasing the problem of obtaining antibiotic-resistant microorganisms. As well as the presence of antibiotic-resistant microorganisms in the larval biomass itself requires their sterilization after separation from the substrate. This issue is not discussed by the authors. Therefore, I consider it premature to conclude that "Collectively, these findings present a novel approach for the management of antibiotic fermentation residue waste through the utilization of BSFL." The paper should necessarily include a discussion or even additional experiments on the problem of obtaining Nosiheptide-resistant bacteria in the BSFL intestine and in the frass. It would be premature to publish an article without this.

Response 1: We thank the reviewer for their positive and supportive comments as well as their constructive criticisms. The transfer of drug‑resistant microorganisms and resistance genes is a critical and challenging issue in antibiotic degradation. The release of antibiotic resistance genes and resistant microorganisms into the environment can cause serious pollution. Initially, this issue was not addressed in the manuscript. A discussion on antibiotic‑resistant bacterial residues has been added in lines 646–659 of the revised manuscript. Furthermore, inhibiting the transfer of antibiotic‑resistant microorganisms and resistance genes is also part of our upcoming experimental plan.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The article is relevant and contains new data on the peculiarities of physiology, metabolism and microbiota of BSF larvae. As presented, it can be published without changes.  

Author Response

Thank you very much for your positive and encouraging evaluation. We are delighted that you find our manuscript highly relevant and consider the new data regarding the physiology, metabolism, and microbiota of black soldier fly larvae to be valuable. Your recommendation to publish the manuscript in its current form without further modification is greatly appreciated. Thank you once again for your valuable time and careful assessment of our work.