Review Reports

Reviewer 1

I accept the manuscript to be published after major revision. the research work is interesting, but some comments must taken in consideration before publication.

Reviewer comments

Authors response

1-N2-adsorption isotherm is required to estimate surface area and sample porosity.

Following Reviewer's suggestion, measurements have been made to determine surface area and porosity of the sorbents tested.

Results of these determinations have been added to sorbents' characteristics (section 3.1).

2-The point of zero charge indicated by author is near 7, so, the solid can attract both cationic and anionic dyes, but why the removal reach maximum at pH=2

As indicated in the manuscript, the sorbents can bind both anionic and cationic dyes. These dyes bind to the sorbent mainly via electrostatic interactions between the ionized functional groups of the sorbent and the sorbate. The sorption effectiveness of these dyes largely depends on the solution pH.

At pH > pH_PZC (pH>8), the sorbents possess a negative charge, which theoretically promotes the sorption of cationic dyes.

In contrast, at pH < pH_PZC (pH < 7), the sorbents possess a positive charge, which enhances the sorption of anionic dyes. The higher the pH value, the more functional groups are ionized (protonated), which results in a greater positive charge and more effective sorption of anionic dyes.

For this reason, dye sorption effectiveness was the highest at pH 2-3 (it has been described more extensively in the revised manuscript).

3-SEM is required only in the revised manuscript to investigate the sample morphology

Mealworm exoskeletons and molts (2-3 mm fraction) are sorptive materials with a highly heterogenous surface.

The morphology, surface structure, and porosity of elements of mealworm exoskeleton derived from abdomen, thorax, head, wing cover (elytra), and legs differ substantially. The same applies to the molts (the surface of which depends on the site they derive from).

Authors are of the opinion that presenting SEM images of elements of mealworm exoskeletons in the manuscript is unnecessary. The number of images would have to be high, while they would not bring much to article's scope.

The study described in the manuscript aimed to investigate the key parameters of sorbents having an impact on dye sorption (chemical nature of sorbent surface - type of functional groups, pHPZC, and total specific surface area and porosity).

SEM images of mealworm exoskeletons and molts are commonly available in the Internet.

Summing up, in authors' opinion, presenting SEM images of mealworm in the manuscript is not necessary.

4-English language must improved in the revised manuscript

English has been checked and corrected as suggested.

5-A stability of catalyst must tested for at lease three consecutive cycles

Authors are not exactly sure what catalyst was meant by the Reviewer. Sorption was not aided with any catalyst. The system included only the sorbent and a dye solution (with pH adjusted using hydrochloric acid).

If the Reviewer meant the re-use of sorbents, it is economically ineffective in our opinion.

Mealworm exoskeletons and molts are waste materials from insect cultures for feeding purposes, hence they are deemed cheap. They prove well as 'single-use' sorbents. Their regeneration is not justified economically.

Regeneration involving dye desorption generates wastewater that needs to be treated/disposed. In addition, it requires using chemical reagents, which generates additional costs.

In authors' opinion, a viable method for managing spent exoskeletons and molts is their drying and co-combustion in, e.g., heating plants, at temperatures > 850oC (energy recovery). Production of activated carbons (via carbonization and activation of biomass of exoskeletons and molts) is also an option.

6-More adsorption equations as Dubinin is better to add to the revised manuscript

Following Reviewer's suggestion, experimental data was described using the Dubinin-Radushkevich model.

Reviewer 2

Just like the manuscript claims, colored wastewater can have a number of negative environmental effects if discharged untreated into natural water bodies, and the sorption process is one of the most attractive options for treating colored effluent. Adsorbents are the core of sorption methods, and the development of inexpensive and easily available adsorbents is very important. The authors proposed mealworm exoskeletons and molts could be successfully used as sorbents to remove dyes from aqueous solutions. These findings were very interesting. However, there were still some problems that need to be improved in the manuscript.

Reviewer comments

Authors response

For example, the manuscript pointed out that pH has a significant influence on the adsorption effect of adsorbent, and the main reason was the influence of surface charge distribution of adsorbent in different pH solutions. To prove the correctness of this viewpoint, the author can add the zeta potential method to measure the charge distribution on the surface of the adsorbent and calculate pHIEP.

Authors are grateful for this suggestion.

The mechanism of pH effect upon the surface charge of the sorbent has been extensively described in literature.

A significant effect of pH on the strength of electrostatic interactions between the sorbent and the sorbate is a common fact. Undoubtedly, a prerequisite for these interactions is the presence of ionizable functional groups in sorbent's and sorbate's structure.

The type of functional groups on the surface of the tested sorbents was identified using FTIR. A ratio between the alkaline and acidic groups on the surface has been estimated based on pH_PZC values. Exact structure of dyes tested in the study and types of functional groups they possess are known.

The significant effect of pH on the surface charge of the sorbents and dye sorption effectiveness has been evidenced in Figure 3 presented in the manuscript.

In authors' opinion, additional proofs supporting this thesis would be redundant and - from reader's perspective - would probably not significantly affect manuscript quality/attractiveness.

The major goal of the study was to demonstrate the usability of exoskeletons and molts of the cultured insect for colored wastewater treatment.

In addition, XPS can be used for further analysis of the surface chemistry of the adsorbent.

In authors' opinion, the study investigated the key parameters of sorbents that affect dye sorption (chemical nature of sorbent surface - type of functional groups, pH_PZC, as well as total specific surface area and porosity).

A precise quantitative analysis of elements made with the XPS spectroscopy method is not necessary in the case of the tested sorbents. During dye sorption, the type of functional groups on sorbent's surface (established based on the FTIR analysis in the present study) is of greater importance than the quantitative composition of elements.

In addition, thorough XPS analysis of the tested sorbents would be very difficult.

Mealworm exoskeletons and molts (2-3 mm fraction) are sorptive materials with a highly heterogenous surface.

The morphology and, presumably, elemental composition of mealworm exoskeleton elements derived from abdomen, thorax, head, wing cover (elytra), and legs differ substantially. The number of necessary XPS analyses would be very high, while they would not bring much to article's scope.

Reviewer 3

The manuscript entitled "The use of exoskeletons and molts of farmed mealworm (Tenebrio molitor) for the removal of reactive dyes from aqueous solutions" aims to test the applicability of exoskeletons and molts from mealworm (Tenebrio molitor) cultures as sorbents for anionic dyes Reactive Black 5 (RB5) and Reactive Yellow (RY84). Factors investigated included characteristics of sorbents (FTIR, pH_PZC), the influence of pH on sorption efficiency, sorption kinetics (models of pseudo-first, pseudo-second-order, intramolecular diffusion), and determination of the maximum sorption capacity (Langmuir 1, Langmuir 2, and Freundlich models). It was a real pleasure to read this manuscript. It is very well prepared. The abstract is suitable. The introduction is informative. The materials and methods are detailed enough. The results presented in this work are excellent and valuable. Moreover, they are carefully shown and thoroughly discussed. The comparison with the literature data is very comprehensive. The conclusions are in line with the presented results. The literature is up-to-date. I have a few minor comments, and they are given below.

Reviewer comments

Authors response

The term "sorbent" is overused in the Introduction. Please, revise.

Following Reviewer's suggestion, the overuse of "sorbent" has been reduced.

The motivation and the novelty of the paper should be clearly stated at the end of the Introduction.

Information about the motivation and the novelty of the paper has been completed in the Introduction section, as suggested by the Reviewer.

The following text has been added:

“…The present work aims to verify the aforementioned hypothesis. To the best of authors' knowledge, fragments of insect exoskeletons have not been investigated as sorbents so far….”

Please, check subtitle 3.2. It is not appropriate.

Authors are grateful for this comment.

Subtitle 3.2 has been corrected.

Figure 3. Please, check the labeling a, b, c, etc. There are errors.

The labeling in Figure 3 has been revised, as suggested.

Line 312: qe – e should be in subscript.

Text has been corrected following Reviewer's suggestion.

Table 3. Since the authors used non-linear forms of adsorption isotherms, it would be useful to include χ2 along with R2 for fitting adequacy assessment.

Following Reviewer's remarks, additional calculations have been performed and χ2 values have been added in Table 3.

The χ2 have also been added in Table 1 (analysis of sorption kinetics).

Reviewer 4

Dear Authors

This manuscript investigates the performance of exoskeletons and molts of mealworm as sorbents for the popular industrial dyes Reactive Black 5 and Reactive Yellow 8. Although, interesting results have been obtained, overall, the manuscript needs major revision before it could be accepted for publication in the Journal of Applied Sciences. In this regard, the author(s) should improve their work according to the following indications.

Reviewer comments

Authors response

1.     Surface morphology (SEM) images should be taken.

Mealworm exoskeletons and molts (2-3 mm fraction) are sorptive materials with a highly heterogenous surface.

The morphology, surface structure, and porosity of elements of mealworm exoskeleton derived from abdomen, thorax, head, wing cover (elytra), and legs differ substantially. The same applies to the molts (the surface of which depends on the site they derive from).

Authors are of the opinion that presenting SEM images of elements of mealworm exoskeletons in the manuscript is unnecessary. The number of images would have to be high, while they would not bring much to article's scope.

The study described in the manuscript aimed to investigate the key parameters of sorbents having an impact on dye sorption (chemical nature of sorbent surface - type of functional groups, pH_PZC, and total specific surface area and porosity).

SEM images of mealworm exoskeletons and molts are commonly available in the Internet.

Summing up, in authors' opinion, presenting SEM images of mealworm in the manuscript is not necessary.

2.     It is required to add a table for comparison of the sorption capacity of different sorbents in relation to contaminants.

Authors are grateful for this remark.

Sorption capacities of various sorbents against RB5 and RY84 dyes analyzed in the present study were compared in Table 4 (presenting a total of 35 various sorbents – literature data).

3.     Explain how variables are selected. What assumptions are made? Explain the considerations

Authors are grateful for this remark.

Analyses of pH effect on dye sorption effectiveness were conducted at a standard pH range, i.e., pH 2-11. The adopted range of pH values enables sorbent testing under both strongly acidic and alkaline conditions.

Analyses of sorption kinetics were carried out at two dye concentrations (50 and 250 mg/L) to allow establishing whether there is a correlation between dye concentration and sorption parameters. The adopted range of concentrations is similar to that of dyes found in colored industrial wastewater.

4.     The sensitivity test should be performed.

A standard research scope was followed in the study.

Each experiment was performed in three replicates. Variability of results is presented in figures in the form of error bars.

The fit of mathematical models to experimental data was analyzed based on determination coefficients (R²) and “chi square” tests (χ²), which is a common and generally accepted practice.

Authors do believe that the adopted research scope and the way the results had been presented are sufficient to demonstrate sorption capabilities of the tested sorbents (and to verify the advanced research hypothesis).

Authors are of the opinion that conducting additional tests (“sensitivity test”) is not necessary in this work.

Such a test is not common in studies of this type.

5.     The optimal operating conditions should be a listed in a Table.

Authors are grateful for this suggestion.

Optimal sorption conditions (pH, equilibrium time) were determined and described in the second and third stage of the study (sections 3.2 and 3.3).

The summary of optimal conditions of dye sorption, convenient to a reader, has been presented in the Conclusion section (which is a common practice).

In authors' opinion, addition of a table that repeats data presented in the manuscript is unnecessary.

6.     Editing for minor grammar corrections is needed.

English has been checked and corrected as suggested.

7.     After literature review, the author should explain better how the paper fill its void. Please clarify the highlights and the novelties of the work.

Following Reviewer's suggestion, the "novelty" of the work and justification of the study have been completed in the Introduction section.

8.     Are the prepared samples recyclable and reusable?

Authors are grateful for this comment.

Mealworm exoskeletons and molts are waste materials from insect cultures for feeding purposes, hence they are deemed cheap. They prove well as 'single-use' sorbents. Their regeneration is rather cost-ineffective.

Regeneration involving dye desorption generates wastewater that needs to be treated/disposed. In addition, it requires using chemical reagents, which generates additional costs.

In authors' opinion, a viable method for managing spent exoskeletons and molts is their drying and co-combustion in, e.g., heating plants, at temperatures > 850^oC (energy recovery). Production of activated carbons (via carbonization and activation of biomass of exoskeletons and molts) is also an option.

9.      The future prospects should be addressed with consideration of the opportunities and challenges.

Authors are very grateful for this suggestion.

The main goals of the study were to determine the sorption capability of mealworm exoskeletons and molts as well as prove the following hypothesis: "the total sorption capacity of mealworm exoskeletons and molts against dyes is greater than the sorption capacity of pure chitin extracted from the same quantity of raw material".

In authors' opinion, these goals have been accomplished.

The manuscript presents a clear suggestion that obtaining chitin from mealworm exoskeletons and molts for sorbent production is economically non-viable.

As suggested by the Reviewer, "the future prospect” will be explored in more detail in future manuscripts.