Biomining of ‘Heavy’ Metals and Lanthanides from Red Mud of a Former Lignite Mines by Sorption on Chitin

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

In this article, the authors examined the ability of chitin as biopolymer to effectively remove aluminum, barium, cadmium, cobalt, copper, manganese, iron, nickel, lead, strontium, and various lanthanides from a red mud-like sludge that originates from the bauxite process, also known as the Bayer process.

The manuscript presents a good research work. In general, it is well organized, with potential to be useful for future research and with the potential for use as in situ treatment of contaminated soils/sediments in the environment.

However, there are some issues that need to be addressed. The manuscript under review cannot be published in this form, so, I recommend major revision.

1.     Specify the novelty in this research.

Response 1: Thank you for pointing this out. We agree partial with this comment. Therefore, we have changed this part to point out the novelty of our research.

Therefore the application of Chitin in biopolymer for different mining options or recycling metals from aqueous sources is widely discussed over the past decade [6,8,11,12,20,22–24]. To the best of our knowledge, there are currently no projects attempting to use chitin as an extraction material for elements from red mud, although there are a few projects exploring the use of red mud as a source for different elements [1,25–29]. As should be evident, the extraction of various metals from red mud is possible using chemical processes involving water. However, the utilization of such processes necessitates the employment of specific chemicals, several of which have been identified as toxic. The objective of the current study is to perform an initial feasibility assessment at the laboratory scale to determine whether it is feasible to utilize chitin to remove various metals from red mud. [Page 4, Line 91 – 102]

Comments 2: At the end of Introduction part explain in more detail objectives of this investigation.

Response 2:  We Agree with this comment and have, changed this part to emphasize this point.

To the best of our knowledge, there are currently no projects attempting to use chitin as an extraction material for elements from red mud, although there are a few projects exploring the use of red mud as a source for different elements [1,25–29]. As should be evident, the extraction of various metals from red mud is possible using chemical processes involving water. However, the utilization of such processes necessitates the employment of specific chemicals, several of which have been identified as toxic. The objective of the current study is to perform an initial feasibility assessment at the laboratory scale to determine whether it is feasible to utilize chitin to remove various metals from red mud. [Page 4, Line 93 – 102]

Comments 3: In 2.1. section, lines-101-103, add a standard method for samples collection.

Response 3: We appreciate the helpful commentary and agree that it might be beneficial to include a standard method here. However, due to the unique characteristics of the stream in question, which is only approximately 45 cm wide and 30 cm deep, we would have to make some adjustments to the method. That's why we've adapted it as described. We're open to incorporating the comment and will try to take it into account by adding a standard method that was carried out in the same region. 

To collect red mud samples from the small stream near a fishpond in Zittau, Germany, a standard method described before by Budelmann [17] is employed. In contrast to the approach proposed by Budelmann, we have adapted the sampling technique to align with local circumstances. The methodology is illustrated below. A glass beaker was submerged in the water near the red mud to obtain samples from the water surface. Ensuring that the samples accurately represented the composition of the red mud present on the surface of the stream. To collect samples from the bottom, a beaker was skillfully driven through the upper layer of the riverbed. By using both methods, we collected a comprehensive and representative set of red mud samples, which were carefully stored in a 1 L sampling glass bottle to preserve their integrity for further analysis. [Page 4, Line 106 – 109]

Comments 4:   Add mass of samples in section 2.3. line 116.

Response 4: First of all, thank you for this helpful comment. However, to help clarify, we have deliberately chosen to show the sample volumes only in Table 1. Due to the different sample volumes, we believe that listing them in the text would be more confusing than listing them in a clear table.

Comments 5: In section 2.4. line 129, describe chitin samples. Are they commercial or not, from which supplier, etc. Also describe the structure of chitin, characteristic functional groups that have the potential for metal binding etc.   

Response 5: We fully agree with the commentary. However, in line with the second reviewer, we think it makes more sense to include this description in the introduction and/or the discussion. We have therefore decided to add only the supplier at this point. We have added a more detailed description of which functional groups are responsible for the metal binding in the introduction.

The dried samples were suspended in either 150 mL DMF (Dimethylformamide)[VWR-Chemicals; P-Code 83634.320] or distilled water, as described in Tabel 1. After stirring all samples at 300 rpm for 30 minutes, 12 g chitin [Sigma-Aldrich; P-Code: 1001295340] were added to the red mud samples, and the reaction flasks were sealed with thin foil. [Page 4-5, Line 128-131]

Chitin, for example, has been shown to bind a wide range of metals, mostly as Me²⁺-Ions [9,13,14,17–21]. As illustrated in Figure 2, chitin is known to contain a variety of potential binding sites for metal ions like Fe²⁺. Fränzle et. al an others [9,17–19] has mainly used chitin for biomonitoring as an alternative to moss monitoring. In this context, the authors have already demonstrated the high binding capability of chitin to various Me²⁺-ions [6,8,9,11,12,17–19,22].

Figure 2 A section of a chitin molecule is presented, along with the potential binding sites for divalent metal ions here using Fe²⁺-Ions as an example.

The high binding capacity of chitin for a variety of metal ions makes it particularly suitable for biomining applications as described by. Therefore the application of Chitin in biopolymer for different mining options or recycling metals from aqueous sources is widely discussed over the past decade [6,8,11,12,20,22–24]. [Page 3-4, Line 81-93]

Comments 6:    In Results and Discussion it would be desirable to add FTIR analysis, i.e. FTIR spectra before and after metal removal, in order to represent functional groups in the chitin structure that are responsible for metal ion binding.

Response 6: We appreciate your point, but unfortunately, we don't have the right equipment to perform an FTIR analysis. However, we've also cited some previous studies that show that the method used here only removes the top layer of chitin from a chitin flake. These studies also indicate that the binding capacity of chitin only decreases after multiple applications. It's possible that individual acetyl groups can be detached by treatment with DMF or formic acid, but we're not sure. However, we don't think this will have a negative effect on the formation behavior. After all, both the free electron pairs of the carbonyl oxygen and the nitrogen of the amino group can complex metal cations. So, we're going to have to refrain from making any changes at this point.

4. Response to Comments on the Quality of English Language

Comments 7:   Several typo’s should be corrected:

˗         On line 125, “Tabel 1“should be corrected to: “Table 1“

˗         On line 127, 2.4. “Desoprtion of ‘hravy’ metals and lanthanides form chitin“, should be corrected to: “2.4. Desorption of ‘heavy’ metals and lanthanides from chitin“.

˗        On line 151 and 152, 3.1. “Compariosn of element concentrations in water with those in the sampled red mud-like sludges“, should be corrected to: “3.1. Comparison of element concentrations in water with those in the sampled red mud-like sludges“.

˗         On line 175, “Measured concentration of the teste elements... “,should be corrected to: “Measured concentration of the tested elements... “.

˗         On line 259, “Biomining of rear earth elements frm red mud-like sludge’s” should be corrected to: “Biomining of rear earth elements from red mud-like sludge’s“.

˗        On lines 219 and 221, “Tabel 2“should be corrected to: “Table 2“.

˗        Also, the Caption for Table 2 should be added.

˗        On line 255, “Figure 4 Recovery rate for the testes elements“should be corrected to: “Figure 4 Recovery rate for the tested elements

Response 7: We fully agree with the comment and have changed the relevant passages in the text. As this is “only” a typographical error, we have taken the liberty of quoting only the relevant passages in the text and not inserting the corresponding passage again

[Page 5, Line 137]

[Page 5, Line 139]

[Page 5, Line 161 and 162]

[Page7, Line 185-187]

[Page10, Line 269]

[Page 8, Line 231]

[Page 10, Line 265-267]

Comments 8:   On line 277, “Tabel 3 Rear earth concentration on chitin of legs from crayfish which life in the teste red mud-like, should be corrected to: “Table 3 Rear earth concentration on chitin of legs from crayfish which life in the tested red mud-like “. Also, below Table 3, on line 279, the caption: “Table 1. This is a table.” is placed. I presume that this is a mistake and should be erased.

Response 8: Thank you for pointing this out, we have changed the relevant passage and corrected the typo. [Page11, Line 287]

5. Additional clarifications

We just wanted to let you know that if there's anything we can clarify or change to make things easier for you, we'd be more than happy to do so.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The study demonstrates the effectiveness of chitin as a sustainable sorbent for biomining heavy metals and lanthanides from red mud, highlights its high adsorption rates and selective metal binding that enhance metal recovery, and broadens the applications of chitin in environmental science, and thus should be considered for publication in the journal.

However, as a carbohydrate chemist, I would appreciate additional data on the structural characterization of chitin before mud treatment and after recovery from mud. For instance, including the degree of N-acetylation of chitin before and after mud treatment would be informative, although not a prerequisite for acceptance of the paper. Given that the mud is rich in NaOH, it is conceivable that the chitin could undergo partial de-N-acetylation due to the high pH treatment.

Response 1: Firstly, it is important to note that we are aware of and agree with your concerns. However, in order to be able to make more precise statements about the degree of N-acetylation, an FTIR analysis or similar would be necessary. Unfortunately, we do not have the necessary hardware for this, which is why we have refrained from such an analysis.

We also understand and share your concerns regarding the potential de-N-acetylation of chitin by NaOH. However, for the following reasons, we hypothesize that this effect may have no or only a minor negative influence on the method described.

1. The complexation of different metal ions occurs via both the carbonyl oxygen and the nitrogen of the amino group. Therefore, partial de-N-acetylation should not have any negative effects.

2. Part of the caustic soda contained in the red mud is usually recycled, which should lower the pH. Furthermore, if red mud is stored above ground, it is subject to various environmental influences, which should result in a further reduction in pH.

Comments 2: The mechanism of metal ion binding to chitin or chitosan should be elaborated in the "Introduction" and/or "Discussion" sections.

Response 2: We fully agree with your comment. For this reason, we have decided to include a corresponding section in the introduction together with a diagram for better illustration.

Chitin, for example, has been shown to bind a wide range of metals, mostly as Me²⁺-Ions [9,13,14,17–21]. As illustrated in Figure 2, chitin is known to contain a variety of potential binding sites for metal ions like Fe²⁺. Fränzle et. al and others [9,17–19] has mainly used chitin for biomonitoring as an alternative to moss monitoring. In this context, the authors have already demonstrated the high binding capability of chitin to various Me²⁺-ions [6,8,9,11,12,17–19,22].

Figure 2 A section of a chitin molecule is presented, along with the potential binding sites for divalent metal ions here using Fe²⁺-Ions as an example.                   [Page 3; Line 81-89]

Comments 3: Figure 2, Figure 3, and Figure 4 appear very raw and need improvement.

Response 3: Although we acknowledge the legitimacy of your concerns, we respectfully disagree with them. The data presented here is not raw data. These values represent the mean of two identical samples, which were previously adjusted by different factors in order to facilitate comparison.

Comments 4: Table 1. “Sample volume” should be changed to “Sample weight”

Response 4: Thank you for pointing this out, we have corrected the spelling mistake as follows.

Table 1 Sample description for adsorption of heavy metals and lanthanides on chitin

[Page 5; Line 137]

Comments 5: There is no title for Table 2.

Response 5: We agree and therefore add a title. We although change Sample Volume in Sample weight according to your Comment 4.

Table 2 Determined total concentration of ions bound to chitin and the resulting saturation rates

[Page 8; Line 131 and 132]

Comments 6: Line 117-118, please add Sigma Cat. No. for DMF and chitin.

Response 6: We fully agree with your comment and have added the relevant information. We have noticed that we do not receive our DMF from Sigma-Aldrich as before, but from VWR-Chemicals, so we have already changed this and added the relevant information.

The dried samples were suspended in either 150 mL DMF (Dimethylformamide)[VWR-Chemicals P-Code 83634.320] or distilled water, as described In Tabel 1. After stirring all samples at 300 rpm for 30 minutes, 12 g chitin [Sigma-Aldrich; P-Code: 1001295340] were added to the red mud samples, and the reaction flasks were sealed with thin foil. [Page 4 and 5; Line 128-131]

Comments 7: Line 1: “Type of the Paper (Article, Review, Communication, etc.)” should be “Article.”

Response 7: We fully agree with your comment and have added Article as the typ of paper [Page 1; Line 1]

Comments 8: Line 2: Capitalize the first letter of each word in the title.

Response 8: We respectfully disagree with your comment. We believe that the capitalization of the first letters of each word in the title is no longer appropriate. This also contravenes the established norms of English spelling, which is why we have deliberately refrained from using this particular spelling.

Comments 9: Line 4: Please add an asterisk (*) to the name of the corresponding author.

Response 9: We fully agree with your comment and have added an asterisk to the name of the corresponding author

[Page 1, Line 4]

Comments 10: Line 78: “Glucosamin” is a typo and should be changed to “Glucosamine.” Lines 77-78: The font format of the letter “D” should be adjusted to “Small caps.”

Response 10: We fully agree with your comments and have amended the corresponding spelling mistakes as follows. We have also included a footnote in which we briefly explain the differences between chitin and chitosan.

Therefore, a cost-effective and user-friendly method is required to eliminate these components from red mud. Biomining using biopolymers such as chitin (ß-1à4 N-acetyl-D-glucosamine)^[1], chitosan (ß-1à4 D-glucosamine)¹, or ‘modified’ cellulose (ß-1à4 D-glucose) is a promising alternative to traditional mining processes.

[Page 3; Line 75-79]

In general, the degree of acetylation of the amino group is used to distinguish between chitin and chitosan. Chitin is distinguished by an acetyl group content above 50%, while chitosan has a lower content below this threshold. It is important to note, however, that native chitin is not typically completely acetylated. This implies that chitin may be a copolymer of D-glucosamine and acetyl-D-glucosamine, as illustrated in Figure 1.  [Page 3]

Comments 11: Line 88: “With M desorption” — What does the letter “M” stand for?

Response 11: We agree with them and have taken care to replace M with Me^X+ ions in our changes.

Comments 12: Line 101: “Researchers” should be changed to “We.”

Response 12: We concur with the comment, but have revised the pertinent section in accordance with the suggestions of the initial reviewer.

To collect red mud samples from the small stream near a fishpond in Zittau, Germany, a standard method described before by Budelmann [17] is employed. In contrast to the approach proposed by Budelmann, we have adapted the sampling technique to align with local circumstances. The methodology is illustrated below.

[Page 4; Line 104-109]

Comments 13: Line 127: “hravy” should be corrected to “heavy.”

Response 13: We agree and change corrected the typo.

2.3. Absoprtion of ‘heavy’ metals and lanthanides on chitin [Page 4; Line 127]

Comments 14: Lines 130, 133, 136: “ml” should be “mL.”

Response 14: We agree with the comment and have adjusted the spelling throughout the document.

Comments 15:

Line 282: “pH value” should be “pH.”

Line 286: “pH-Value” should be “pH.”

Please change all instances of “pH value” to “pH.”

Response 15: We agree with the comment and have adjusted the spelling throughout the document.

4. Response to Comments on the Quality of English Language

Point 1: The English language throughout the document requires thorough polishing before it can be considered for publication.

Response 1: First of all, we would like to thank you for your advice on how to improve our language. After another review by the second author, Prof. Dr. Fränzle, we were able to correct the grammatical and linguistic problems in our opinion. As a result, we believe that the text should now be linguistically better than before. Of course, we will be happy to improve it further if you still find it insufficient. For now, however, we believe that the linguistic issues should be resolved.

Due to the many minor spelling and grammatical errors throughout the document, we have decided not to list each change here, and instead refer you to the revised text. The changed passages are highlighted there.

5. Additional clarifications

We just wanted to let you know that if there's anything we can clarify or change to make things easier for you, we'd be more than happy to do so.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The study demonstrates the effectiveness of chitin as a sustainable sorbent for biomining heavy metals and lanthanides from red mud, highlights its high adsorption rates and selective metal binding that enhance metal recovery, and broadens the applications of chitin in environmental science, and thus should be considered for publication in the journal.

However, as a carbohydrate chemist, I would appreciate additional data on the structural characterization of chitin before mud treatment and after recovery from mud. For instance, including the degree of N-acetylation of chitin before and after mud treatment would be informative, although not a prerequisite for acceptance of the paper. Given that the mud is rich in NaOH, it is conceivable that the chitin could undergo partial de-N-acetylation due to the high pH treatment.

Response 1: Firstly, it is important to note that we are aware of and agree with your concerns. However, in order to be able to make more precise statements about the degree of N-acetylation, an FTIR analysis or similar would be necessary. Unfortunately, we do not have the necessary hardware for this, which is why we have refrained from such an analysis.

We also understand and share your concerns regarding the potential de-N-acetylation of chitin by NaOH. However, for the following reasons, we hypothesize that this effect may have no or only a minor negative influence on the method described.

1. The complexation of different metal ions occurs via both the carbonyl oxygen and the nitrogen of the amino group. Therefore, partial de-N-acetylation should not have any negative effects.

2. Part of the caustic soda contained in the red mud is usually recycled, which should lower the pH. Furthermore, if red mud is stored above ground, it is subject to various environmental influences, which should result in a further reduction in pH.

Comments 2: The mechanism of metal ion binding to chitin or chitosan should be elaborated in the "Introduction" and/or "Discussion" sections.

Response 2: We fully agree with your comment. For this reason, we have decided to include a corresponding section in the introduction together with a diagram for better illustration.

Chitin, for example, has been shown to bind a wide range of metals, mostly as Me²⁺-Ions [9,13,14,17–21]. As illustrated in Figure 2, chitin is known to contain a variety of potential binding sites for metal ions like Fe²⁺. Fränzle et. al and others [9,17–19] has mainly used chitin for biomonitoring as an alternative to moss monitoring. In this context, the authors have already demonstrated the high binding capability of chitin to various Me²⁺-ions [6,8,9,11,12,17–19,22].

Figure 2 A section of a chitin molecule is presented, along with the potential binding sites for divalent metal ions here using Fe²⁺-Ions as an example.                   [Page 3; Line 81-89]

Comments 3: Figure 2, Figure 3, and Figure 4 appear very raw and need improvement.

Response 3: Although we acknowledge the legitimacy of your concerns, we respectfully disagree with them. The data presented here is not raw data. These values represent the mean of two identical samples, which were previously adjusted by different factors in order to facilitate comparison.

Comments 4: Table 1. “Sample volume” should be changed to “Sample weight”

Response 4: Thank you for pointing this out, we have corrected the spelling mistake as follows.

Table 1 Sample description for adsorption of heavy metals and lanthanides on chitin

[Page 5; Line 137]

Comments 5: There is no title for Table 2.

Response 5: We agree and therefore add a title. We although change Sample Volume in Sample weight according to your Comment 4.

Table 2 Determined total concentration of ions bound to chitin and the resulting saturation rates

[Page 8; Line 131 and 132]

Comments 6: Line 117-118, please add Sigma Cat. No. for DMF and chitin.

Response 6: We fully agree with your comment and have added the relevant information. We have noticed that we do not receive our DMF from Sigma-Aldrich as before, but from VWR-Chemicals, so we have already changed this and added the relevant information.

The dried samples were suspended in either 150 mL DMF (Dimethylformamide)[VWR-Chemicals P-Code 83634.320] or distilled water, as described In Tabel 1. After stirring all samples at 300 rpm for 30 minutes, 12 g chitin [Sigma-Aldrich; P-Code: 1001295340] were added to the red mud samples, and the reaction flasks were sealed with thin foil. [Page 4 and 5; Line 128-131]

Comments 7: Line 1: “Type of the Paper (Article, Review, Communication, etc.)” should be “Article.”

Response 7: We fully agree with your comment and have added Article as the typ of paper [Page 1; Line 1]

Comments 8: Line 2: Capitalize the first letter of each word in the title.

Response 8: We respectfully disagree with your comment. We believe that the capitalization of the first letters of each word in the title is no longer appropriate. This also contravenes the established norms of English spelling, which is why we have deliberately refrained from using this particular spelling.

Comments 9: Line 4: Please add an asterisk (*) to the name of the corresponding author.

Response 9: We fully agree with your comment and have added an asterisk to the name of the corresponding author

[Page 1, Line 4]

Comments 10: Line 78: “Glucosamin” is a typo and should be changed to “Glucosamine.” Lines 77-78: The font format of the letter “D” should be adjusted to “Small caps.”

Response 10: We fully agree with your comments and have amended the corresponding spelling mistakes as follows. We have also included a footnote in which we briefly explain the differences between chitin and chitosan.

Therefore, a cost-effective and user-friendly method is required to eliminate these components from red mud. Biomining using biopolymers such as chitin (ß-1à4 N-acetyl-D-glucosamine)^[1], chitosan (ß-1à4 D-glucosamine)¹, or ‘modified’ cellulose (ß-1à4 D-glucose) is a promising alternative to traditional mining processes.

[Page 3; Line 75-79]

In general, the degree of acetylation of the amino group is used to distinguish between chitin and chitosan. Chitin is distinguished by an acetyl group content above 50%, while chitosan has a lower content below this threshold. It is important to note, however, that native chitin is not typically completely acetylated. This implies that chitin may be a copolymer of D-glucosamine and acetyl-D-glucosamine, as illustrated in Figure 1.  [Page 3]

Comments 11: Line 88: “With M desorption” — What does the letter “M” stand for?

Response 11: We agree with them and have taken care to replace M with Me^X+ ions in our changes.

Comments 12: Line 101: “Researchers” should be changed to “We.”

Response 12: We concur with the comment, but have revised the pertinent section in accordance with the suggestions of the initial reviewer.

To collect red mud samples from the small stream near a fishpond in Zittau, Germany, a standard method described before by Budelmann [17] is employed. In contrast to the approach proposed by Budelmann, we have adapted the sampling technique to align with local circumstances. The methodology is illustrated below.

[Page 4; Line 104-109]

Comments 13: Line 127: “hravy” should be corrected to “heavy.”

Response 13: We agree and change corrected the typo.

2.3. Absoprtion of ‘heavy’ metals and lanthanides on chitin [Page 4; Line 127]

Comments 14: Lines 130, 133, 136: “ml” should be “mL.”

Response 14: We agree with the comment and have adjusted the spelling throughout the document.

Comments 15:

Line 282: “pH value” should be “pH.”

Line 286: “pH-Value” should be “pH.”

Please change all instances of “pH value” to “pH.”

Response 15: We agree with the comment and have adjusted the spelling throughout the document.

4. Response to Comments on the Quality of English Language

Point 1: The English language throughout the document requires thorough polishing before it can be considered for publication.

Response 1: First of all, we would like to thank you for your advice on how to improve our language. After another review by the second author, Prof. Dr. Fränzle, we were able to correct the grammatical and linguistic problems in our opinion. As a result, we believe that the text should now be linguistically better than before. Of course, we will be happy to improve it further if you still find it insufficient. For now, however, we believe that the linguistic issues should be resolved.

Due to the many minor spelling and grammatical errors throughout the document, we have decided not to list each change here, and instead refer you to the revised text. The changed passages are highlighted there.

5. Additional clarifications

We just wanted to let you know that if there's anything we can clarify or change to make things easier for you, we'd be more than happy to do so.