Preparation of Chitosan-Diatomite/Calcium Alginate Composite Hydrogel Beads for the Adsorption of Congo Red Dye

Round 1

Reviewer 1 Report

In this manuscript, the authors reported "Preparation of chitosan-diatomite/sodium alginate composite hydrogel beads for the adsorption of Congo red dye". The work is interesting. I recommend it be accepted for publication after some revising. The main concerns are as follows.

1.     In the abstract: The influence of contact time, solution pH, initial dye concentration, amount of CS-DE@CA, and solution temperature results should be included.

2.     Why did the authors choose Congo red dye for the present study?

3.     The authors should give justification to prove the novelty of this research.

4.     Add the purity of the starting materials.

5.     In Figure 2: The authors should include the EDAX analysis of the CS-DE@CA.

6.     The authors must add the XRD analysis of CS-DE@CA.

7.     The authors should calculate thermodynamic parameters such as Gibbs free energy, entropy change, and enthalpy change.

8.     The authors need to compare the present thermodynamic parameters with previously published results in tabular form.

9.     The authors should discuss the adsorption mechanism in detail and include the before and after dye adsorption FT-IR or SEM analyses.    

10.  The novelty should be highlighted in the conclusion.

11.  Enhance the all-figure qualities.  

12.  Improve the figure captions and add more details.

13.  There are more typos in the manuscript; double-check it thoroughly.

The English language is good. 

Author Response

1. In the abstract: The influence of contact time, solution pH, initial dye concentration, amount of CS-DE@CA, and solution temperature results should be included.

Response: Thank you very much for your valuable comments. We have added these information in the main text according to the your suggestions, and we have also maked table and list of some information, which are helpful for us.

2. Why did the authors choose Congo red dye for the present study?

Response: Of course, its degradation by-product under anaerobic circumstance is benzidine, which is harmful and toxic to human organs and tissue.

3. The authors should give justification to prove the novelty of this research.
4. Add the purity of the starting materials.
5. In Figure 2: The authors should include the EDAX analysis of the CS-DE@CA.
6. The authors must add the XRD analysis of CS-DE@CA.

Response: We are grateful to your positive comments and kind suggestions, which are very important to improve the quality of our manuscript. In the revised manuscript, we have also added another experiment and the corresponding sentences.

7. The authors should calculate thermodynamic parameters such as Gibbs free energy, entropy change, and enthalpy change.
8. The authors need to compare the present thermodynamic parameters with previously published results in tabular form.

Response: Thank you very much for your kindly suggestion. Due to the present difficulty in our group, the thermodynamic experiment can not come true, we will try to let this experiment come true in the next coming year.

9. The authors should discuss the adsorption mechanism in detail and include the before and after dye adsorption FT-IR or SEM analyses.  
10. The novelty should be highlighted in the conclusion.
11. Enhance the all-figure qualities.  
12. Improve the figure captions and add more details.
13. There are more typos in the manuscript; double-check it thoroughly.

Response:We appreciate to your helpful comments and kind suggestions, which are very important to improve the quality of our manuscript. According to your comment, English expression have been revised carefully, figure labellig, units and abbreviations in the original manuscript have been also revised in the revised manuscript. and the involved portions have been marked in red in the revised manuscript.

Reviewer 2 Report

In the final sorbent composition context, the title should be changed into [...] chitosan-diatomite/calcium alginate [...]

Compare the current results whit the sorption of the Congo red on the CS, CA matrix (if available in the literature). It will be nice to prove that the dye sorption is connected with the presence of DE and is not only a result of the sorption by the polymers used. It was mentioned in the methodology section (line 102) that CA and DE@CA beds were also formed. Why are there no sorption results on these beads?
Is there any proof that DE also adsorbed the Congo red?

While discussing the mechanism, refer to the proposed internal structure /confirmed by SEM imaging/. This structure should be mentioned in the section devoted to SEM analysis.

Line 15 - the values indicate that the adsorption capacity increased
Lines 40-52: are there any research devoted to the sorption of congo red by neat diatomite? Please refer to the capacity of the sorbent in the dye removal process
Line 69: NH₂ functional groups (they become NH₃⁺ functionals after protonation)
Line 72: change Scheme.1. into Scheme 1
Introduction: Please explain why DE needs to be coated with chitosan and why DE can not be entrapped within the Ca²⁺ crosslinked alginate matrix.
Line 82: "chloridewere"
Materials: please provide the characteristics of polymers used (molecular weight and degree of deacetylation (DD) of chitosan, the molecular weight of alginate, as these features - especially DD - affects the final sorption capacity and material structure)
Line 91: Please indicate if the dispersion of DE in the CS solution was formed. Moreover, was the mixture stirred or sonicated?
Line 92: provide the concentration of the acetic acid solution
Line 96: give the solvent (water?)
Line 99: 2 wt.%
Line 99: provide the needle size if such a system was used (in the other case, give the size/volume of the drops)
Line 105: provide the procedure of SEM analysis (Au/Pt coated?), give the atmosphere (air/nitrogen) for TG measurements, and FTIR analysis provide the method (powdered samples in the KBr-disc form?)
Line 112: congo red aqueous solutions?
Line 114: why different dyes if only one type was suggested in the introduction
Line 110: the description of sorption and desorption is laconic: provide the mass of the adsorbent or mass/volume ratio, and provide the contact time; for the isotherm studies, the constant temperature should be provided (please give the temperature used in this study), time of the resorption in sodium hydroxide solution, 
Line 140: bands at 2928 and 2885 belong to symmetric and antisymmetric CH₂ vibrations of carbohydrate ring (not OH) - please see the FTIR analysis of neat polymers provided, e.g., Carbohydrate Polymers 77 (2009) 590–598
Lines 131-149: please refer to the chemical structure of alginate, what band confirms the presence of this polymer. Moreover, alginate and chitosan can interact with each other through the electrostatic interaction of NH3+ and COO-. Is there any confirmation that the chitosan layer ionically bounded the alginate layer?
SEM images: What interactions are responsible for the stability of the CS and DE interactions? The CA layer covering the DE@CS is dense or porous?
Figure 3: provide the DTG curves and present the stage parameters in the table (onset temperature, weight loss). Explain why the CS-DE@CA data are compared with CA and not with CS-DE and DE. What should the TG analysis show?
Line 132: it can be seen that the second stage corresponds starts at temperatures lower than 300 (probably 300 is the moment representing the fastest weight loss)
Figure 4: the X axis should be denoted as "t" not "T" (suggesting temperature)
Lines 213 and 214: time of the sorption process should be mentioned in comparison (after  .... h)
Line 216: at pH=7 only some NH₂ functionals are protonated. According to the pKa value ionization degree is ca. 25%. If chitosan is easily protonated in pH=7, it will be soluble in water.
Line 219: how can it be bound by the NH₂ on the surface if the surface constitutes the calcium alginate layer?
Line 231: the mentioned values represent a given sorption time (please add time in this sentence)
Figure 6b - add unit (C_e/q_e)
Line 274: change "better" to "higher"
Line 316-317: here, the possible interaction between protonated amino groups of Congo red and negatively charged functionals of alginate are mentioned, as a reason for the sorption, while earlier, the interactions with chitosan were suggested. Please describe all possible interactions, as the sorption process can be a complex phenomenon in the case of multi-component materials.
Lines 329-330: I did not find proof that DE is mainly responsible for a high sorption efficiency - primarily as the sorption process was found to be chemical (characteristic for polymer-dye interactions) rather than physical (characteristic for DE).

minor changes required

Author Response

Thanks for the positive comments and the instructive questions of the reviewers. The manuscript has been revised according to the comments. The content of the responses to the comments have been incorporated in the revised manuscript. The revised portions are marked in red in the revised manuscript.

Compare the current results whit the sorption of the Congo red on the CS, CA matrix (if available in the literature). It will be nice to prove that the dye sorption is connected with the presence of DE and is not only a result of the sorption by the polymers used. It was mentioned in the methodology section (line 102) that CA and DE@CA beds were also formed. Why are there no sorption results on these beads?
Is there any proof that DE also adsorbed the Congo red?

Response: Thank you very much for your kindly suggestion. We have carefully searched the relevant research works based on the adsorption of diatomite (DE) for Congo red by different groups, and we have introduced their work in the introduction section, which are shown as,

Vishal et. al. reported that the low-cost diatomite with Zn-Mg-Al Layered triple hydroxide (LTH) adsorbents for the effectual removal of Congo red.(DOI: 10.1016/j.colsurfa.2023.130922). A bare DE (20 mg) showed a removal efficiency of only 7.4% at pH 7. This indicates that DE also adsorbed the Congo red. CA beads was used for TGA test. Compared to the removal efficiencies of CS-DE@CA beads, DE@CA beds showed the lower removal efficiencies (⁓83.6 %).

While discussing the mechanism, refer to the proposed internal structure /confirmed by SEM imaging/. This structure should be mentioned in the section devoted to SEM analysis.

Response: We are grateful to your positive comments and kind suggestions, which are very important to improve the quality of our manuscript. In the revised manuscript, we have also added another experiment and the corresponding sentences.

Line 15 - the values indicate that the adsorption capacity increased
Lines 40-52: are there any research devoted to the sorption of congo red by neat diatomite? Please refer to the capacity of the sorbent in the dye removal process
Line 69: NH2 functional groups (they become NH3+ functionals after protonation)
Line 72: change Scheme.1. into Scheme 1
Introduction: Please explain why DE needs to be coated with chitosan and why DE can not be entrapped within the Ca2+ crosslinked alginate matrix.
Line 82: "chloridewere"
Materials: please provide the characteristics of polymers used (molecular weight and degree of deacetylation (DD) of chitosan, the molecular weight of alginate, as these features - especially DD - affects the final sorption capacity and material structure)
Line 91: Please indicate if the dispersion of DE in the CS solution was formed. Moreover, was the mixture stirred or sonicated?
Line 92: provide the concentration of the acetic acid solution
Line 96: give the solvent (water?)
Line 99: 2 wt.%
Line 99: provide the needle size if such a system was used (in the other case, give the size/volume of the drops)
Line 105: provide the procedure of SEM analysis (Au/Pt coated?), give the atmosphere (air/nitrogen) for TG measurements, and FTIR analysis provide the method (powdered samples in the KBr-disc form?)
Line 112: congo red aqueous solutions?
Line 114: why different dyes if only one type was suggested in the introduction
Line 110: the description of sorption and desorption is laconic: provide the mass of the adsorbent or mass/volume ratio, and provide the contact time; for the isotherm studies, the constant temperature should be provided (please give the temperature used in this study), time of the resorption in sodium hydroxide solution, 
Line 140: bands at 2928 and 2885 belong to symmetric and antisymmetric CH2 vibrations of carbohydrate ring (not OH) - please see the FTIR analysis of neat polymers provided, e.g., Carbohydrate Polymers 77 (2009) 590–598
Lines 131-149: please refer to the chemical structure of alginate, what band confirms the presence of this polymer. Moreover, alginate and chitosan can interact with each other through the electrostatic interaction of NH3+ and COO-. Is there any confirmation that the chitosan layer ionically bounded the alginate layer?
SEM images: What interactions are responsible for the stability of the CS and DE interactions? The CA layer covering the DE@CS is dense or porous?
Figure 3: provide the DTG curves and present the stage parameters in the table (onset temperature, weight loss). Explain why the CS-DE@CA data are compared with CA and not with CS-DE and DE. What should the TG analysis show?
Line 132: it can be seen that the second stage corresponds starts at temperatures lower than 300 (probably 300 is the moment representing the fastest weight loss)
Figure 4: the X axis should be denoted as "t" not "T" (suggesting temperature)
Lines 213 and 214: time of the sorption process should be mentioned in comparison (after  .... h)
Line 216: at pH=7 only some NH2 functionals are protonated. According to the pKa value ionization degree is ca. 25%. If chitosan is easily protonated in pH=7, it will be soluble in water.
Line 219: how can it be bound by the NH2 on the surface if the surface constitutes the calcium alginate layer?
Line 231: the mentioned values represent a given sorption time (please add time in this sentence)
Figure 6b - add unit (Ce/qe)
Line 274: change "better" to "higher"
Line 316-317: here, the possible interaction between protonated amino groups of Congo red and negatively charged functionals of alginate are mentioned, as a reason for the sorption, while earlier, the interactions with chitosan were suggested. Please describe all possible interactions, as the sorption process can be a complex phenomenon in the case of multi-component materials.
Lines 329-330: I did not find proof that DE is mainly responsible for a high sorption efficiency - primarily as the sorption process was found to be chemical (characteristic for polymer-dye interactions) rather than physical (characteristic for DE).

Response: Thank you very much for your valuable comments. We have added these information in the main text according to the your suggestions.

Round 2

Reviewer 2 Report

I didn't find the changes or comments to several of my previous suggestions:
1. In the final sorbent composition context, the title should be changed into [...] chitosan-diatomite/calcium alginate [...]
2. There is still no proof that the dye sorption is connected with the presence of DE and is not only a result of the sorption by the polymers.
3. It was mentioned in the methodology section (line 109) that CA and DE@CA beds were also formed. Why are there no sorption results on these beads?
4. Improve the discussion of the sorption mechanism; refer to the proposed internal structure /confirmed by SEM imaging/. This structure should be mentioned in the section devoted to SEM analysis.
5. Line 16-18: "It was also found that the adsorption capacity of Congo Red decreased from 23.28 mg/g to 38.84 mg/g (increased?) when the starting concentration increased from 25 17 mg/L to 75 mg/L."
6. Line 71: “CS is the presence of large amount of NH2+ groups (???) that can be protonated,”: NH2 functional groups (they become NH3+ functionals after protonation)
7. Line 75: change Scheme.1. into Scheme 1
8. Introduction: Please explain why DE needs to be coated with chitosan and why DE can not be entrapped within the Ca²⁺ crosslinked alginate matrix.
9. Materials: please provide the characteristics of polymers used (molecular weight)
10. Line 124: give the solvent for preparation of congo red solutions
11. FTIR: please refer to the chemical structure of alginate, what band confirms the presence of this polymer. Moreover, alginate and chitosan can interact with each other through the electrostatic interaction of NH₃⁺ and COO^-. Is there any confirmation that the chitosan layer ionically bounded the alginate layer? The discussion regarding the possible interactions between chitosan, alginate and Ca(II) is laconic, but it is crucial for the sorption, as the crosslinking process affects the available sorption sites of the sorbent.
12. Line 203-204: if the aim of the TG analysis was to confirm the effect of the thermal stability of alginate on the thermal stability of the final CS-DE@CA thus it should be rather compared with uncoated CS-DE in the context of CA rather than only CA.
13. Line 245-247: how can it be bound by the NH₂ on the surface if the surface constitutes the calcium alginate layer?
14. Line 259-260: the mentioned values represent a given sorption time (please add time in this sentence)
15. Figure 6b - add unit (Ce/qe), as q is (mg/g), while C is expressed as (mg/L)
16. Conclusions: I did not find proof that DE is mainly responsible for a high sorption efficiency - primarily as the sorption process was found to be chemical (characteristic for polymer-dye interactions) rather than physical (characteristic for DE).

-

Author Response

I didn't find the changes or comments to several of my previous suggestions:
1. In the final sorbent composition context, the title should be changed into [...] chitosan-diatomite/calcium alginate [...]

Response: Thank you very much for your kind support. We also sincerely thank you for your comments and suggestions on our work during the review process. These valuable comments will be hugely beneficial for our future research. In the final sorbent composition context, we have already change to chitosan-diatomite/calcium alginate.
2. There is still no proof that the dye sorption is connected with the presence of DE and is not only a result of the sorption by the polymers.

Response: Thank you very much for your kind suggestion, In the Line 51, we added the result that the maximum adsorption capacity of diatomaceous earth and the modified diatomaceous earth for Congo red.
3. It was mentioned in the methodology section (line 109) that CA and DE@CA beds were also formed. Why are there no sorption results on these beads?

Response: Thank you very much for your kind suggestion,we are sorry that our work only tested DE@CA beads removal rate in the line 246. As a comparison, DE@CA had the lower removal efficiencies (⁓83.6 %) and CS-DE@CA had the higher removal efficiencies (⁓89.9 %) after 180 h.
4. Improve the discussion of the sorption mechanism; refer to the proposed internal structure /confirmed by SEM imaging/. This structure should be mentioned in the section devoted to SEM analysis.

Response: The content of the responses to the comments have been incorporated in the revised manuscript. The revised portions are marked in red in the line 185-186.
5. Line 16-18: "It was also found that the adsorption capacity of Congo Red decreased from 23.28 mg/g to 38.84 mg/g (increased?) when the starting concentration increased from 25 17 mg/L to 75 mg/L."

Response: Thank you very much for your kind suggestion. We have made modifications and marked them in red.
6. Line 71: “CS is the presence of large amount of NH2+ groups (???) that can be protonated,”: NH2 functional groups (they become NH3+ functionals after protonation)

Response: Thank you very much for your kind suggestion. We have been made corresponding modifications and marked them in red.
7. Line 75: change Scheme.1. into Scheme 1
8. Introduction: Please explain why DE needs to be coated with chitosan and why DE can not be entrapped within the Ca2+ crosslinked alginate matrix.

Response: By chitosan-coated with DE, the adsorption performance of powder material DE can be improved, We have been made corresponding sentence(line 74-77) and marked them in red.
9. Materials: please provide the characteristics of polymers used (molecular weight)

Response: Thank you very much for your kind suggestion. The instruction of sodium alginate (we used) has not molecular weight（Image in attachment）. We added other characteristics of sodium alginate,such as: (Na-alginate, loss on drying ≤ 22%).
10. Line 124: give the solvent for preparation of congo red solutions

Response: Thank you very much for your kind suggestion. We have been made corresponding modifications and marked them in red.
11. FTIR: please refer to the chemical structure of alginate, what band confirms the presence of this polymer. Moreover, alginate and chitosan can interact with each other through the electrostatic interaction of NH3+ and COO-. Is there any confirmation that the chitosan layer ionically bounded the alginate layer? The discussion regarding the possible interactions between chitosan, alginate and Ca(II) is laconic, but it is crucial for the sorption, as the crosslinking process affects the available sorption sites of the sorbent.

Response: We are grateful to your positive comments and kind suggestions. We have been add the corresponding sentence (line 182-188).
12. Line 203-204: if the aim of the TG analysis was to confirm the effect of the thermal stability of alginate on the thermal stability of the final CS-DE@CA thus it should be rather compared with uncoated CS-DE in the context of CA rather than only CA.

Response: We are grateful to your positive comments and kind suggestions. We have been made corresponding modifications and marked them in red.

13. Line 245-247: how can it be bound by the NH2 on the surface if the surface constitutes the calcium alginate layer?

Response: With the aid of the crosslinking of divalent cations, SA can form stable hydrogel, a 3D flexible polymeric network, which exhibits divergent swelling due to various ratios of hydrophilic to hydrophobic functional groups.（10.1016/j.matchemphys.2020.123931）
14. Line 259-260: the mentioned values represent a given sorption time (please add time in this sentence)
15. Figure 6b - add unit (Ce/qe), as q is (mg/g), while C is expressed as (mg/L)

Response:We have been add time and and unit (Ce/qe)，marked them in red.
16. Conclusions: I did not find proof that DE is mainly responsible for a high sorption efficiency - primarily as the sorption process was found to be chemical (characteristic for polymer-dye interactions) rather than physical (characteristic for DE).

Response:We are grateful to your positive comments and kind suggestions, which are very important to improve the quality of our manuscript. In the conclusions, we have also added the corresponding sentences.

Author Response File: Author Response.pdf