The Important Role of Dissolved Oxygen Supply Regulated by the Hydraulic Shear Force during the Biosynthesis of Iron Hydroxysulfate Minerals

Round 1

Reviewer 1 Report

1. Lines 298-299 “At low Fe²⁺ oxidation efficiency, the hydrolytic mineralization product of Fe³⁺ is mainly schwertmannite, while high Fe²⁺ oxidation efficiency promotes jarosite formation.” Could you explain why does it happened?
2. Have you considered the role of extracellular polymeric substances in the biomineral precipitation?
3. Have you measured the Fe³⁺ concentration and pH changes during biomineralization?
4. The morphology of the crystals should be compared to literature data.
5. It could be interesting to give the content of the minerals using Rietveld refinement as well as the crystallites’ size (based on XRD data).
6. There is no discussion part in the manuscript. It should be added or section Results should be extended on the discussion of the results with literature data.

Author Response

Point-by-point responses to the reviewers’ comments

Ms. Ref. No.: minerals-782178
Title: The important role of dissolved oxygen supply regulated by hydraulic shear force in the biosynthesis of iron hydroxysulfate minerals

Authors: Jun Yang, Rui Wang, Heru Wang, Zhenyu Wang and Yongwei Song *

 

Reviewer #1:

Major concerns:

Comment 1: Lines 298-299 “At low Fe²⁺ oxidation efficiency, the hydrolytic mineralization product of Fe³⁺ is mainly schwertmannite, while high Fe²⁺ oxidation efficiency promotes jarosite formation.” Could you explain why does it happened?

Response:

Thank you for your comments. Bai et al. (2012) observed that either amorphous schwertmannite or crystalline jarosite existed at the critical point of jarosite formation in FeSO₄-K₂SO₄-H₂O system by A. ferrooxidans, depended on the rate of Fe³⁺ supply. The lower supply rate of Fe³⁺ obviously inhibited K⁺ incorporation into iron hydroxysulfate mineral to form jarosite, consequently improved the generation of amorphous schwertmannite in the system (Fig. 1). Meanwhile, the influence of initial Fe²⁺ concentration on the mineralogy of the precipitates formed in solutions containing 4 mmol/L of K⁺ was investigated. As shown in Fig. 2, the precipitate was the pure schwertmannite when the initial Fe²⁺ concentrations was below 40 mmol/L, while the secondary ferric minerals were mixtures of schwertmannite and jarosite when theinitial Fe²⁺ concentrations was above 80 mmol/L. In other words, at a fixed K⁺ concentration, lower initial Fe²⁺ concentration did not lead to the formation of jarosite. These results implied that the crystallinity of the secondary ferric minerals increased with the increase of initial Fe²⁺ concentration in the medium with a fixed K⁺ concentration. In order to make the readers better understand the reason, we explained it in the revised manuscript.

Fig. 1

Fig. 2

Bai, S.Y.; Xu, Z.H.; Wang, M.; Liao, Y.H.; Liang, J.R.; Zheng, C.C.; Zhou, L.X. Both initial concentrations of Fe(II) and monovalent cations jointly determine the formation of biogenic iron hydroxysulfate precipitates in acidic sulfate-rich environments. Mat. Sci. Eng. C 2012, 32, 2323–2329.

 

Comment 2: Have you considered the role of extracellular polymeric substances in the biomineral precipitation?

Response:

The reviewer's analysis is very good, which indicates that the reviewer has deep knowledge in the field of iron hydroxysulfate minerals. In fact, we have investigated the differences between the chemical (H₂O₂-FeSO₄) and biological methods (A. ferrooxidans-FeSO₄) for the synthesis of schwertmannite at the same Fe²⁺ oxidation efficiency (Song et al., 2018). The results indicated that the chemical method is more conducive to the synthesis of schwertmannite than the biological method. The biosynthesized schwertmannite displayed “chestnut shell” or “sea urchin” particle shapes, with a particle median diameter of ~1.52 µm and a specific surface area of 58.79 m²/g. In contrast, the surface of the chemically synthesized schwertmannite was relatively smooth, with a particle median diameter of ~1.34 µm and a specific surface area of only 6.31 m²/g (Fig. 3). In other words, the synthesis of schwertmannite is not only affected by the efficiency of Fe²⁺ oxidation, but also by the characteristics of A. ferrooxidans. After the stripping of the extracellular polymeric substances (EPS) from A. ferrooxidans by high-speed centrifugation, the biomineralized schwertmannite changed from “chestnut shell” or “sea urchin” to “wool ball” or “sponge” particle shapes, with a larger specific surface area of 86.43 m²/g (Fig. 4). Meanwhile, the yield of schwertmannite catalytically synthesized by A. ferrooxidans increased by about 45% compared with that before stripping.

Fig. 3

Fig. 4

Song, Y.W.; Liu, Y.L.; Wang, H.R. Comparison of the biological and chemical synthesis of schwertmannite at a consistent Fe²⁺ oxidation efficiency and the effect of extracellular polymeric substances of Acidithiobacillus ferrooxidans on biomineralization. Materials, 2018, 11(9): DOI: 10.3390/ma11091739.

 

Comment 3: Have you measured the Fe³⁺ concentration and pH changes during biomineralization?

Response:

Yes. The pH values of the solutions were determined by a pHS-3C precision pH meter. The Fe²⁺ and total Fe (TFe) concentrations were determined using the 1,10-phenanthroline method by a spectrophotometer (Liu et al., 2015), and the concentration of Fe³⁺ was calculated as follows: Fe³⁺=TFe-Fe²⁺. This study focuses on the effect of hydraulic shear force on the biological oxidation of Fe²⁺ and the mineralization ability of TFe, so the pH value and Fe³⁺ concentration are not shown in this paper.

Liu, F.W.; Gao. S.Y.; Wang, M.; Yu, H.Y.; Cui, C.H.; Zhou, L.X. Effect of KOH on the formation of biogenic secondary iron minerals in iron- and sulfate-rich acidic environment. Acta Scien. Circum. 2015, 35, 476–483.

 

Comment 4: The morphology of the crystals should be compared to literature data.

Response:

Thank you for your comments. According to the reviewer's suggestion, we have already cited relevant literatures in the revised manuscript (Sasaki and Konno 2000; Loan et al. 2005; Gramp et al. 2008; Liu et al. 2013). Please refer to the revised manuscript (marked in red).

Sasaki, K.; Konno, H. Morphology of jarosite-group compounds precipitated from biologically and chemically oxidized Fe ions. The Canadian mineralogist 2000, 38, 45–56.

Loan, M.; Richmond, W.R.; Parkinson, G.M. On the crystal growth of nanoscale schwertmannite. J. Cryst. Growth 2005, 275, 1875–1881.

Gramp, J.P.; Sandy, J.F.; Bigham, J.M.; Tuovinen, O.H. Monovalent cation concentrations determine the types of Fe(III) hydroxysulfate precipitates formed in bioleach solutions. Hydrometallurgy 2008, 94, 29–33.

Liu, F.W.; Bu, Y.S.; Tian, G.J.; Cui, C.H.; Zhou, L.X. Influence of temperature and pH on dissolution behavior of biogenic Schwertmannite in acidic environment and the adsorption of Cu²⁺. Acta Scien. Circum. 2013, 33, 2445–2451.

 

Comment 5: It could be interesting to give the content of the minerals using Rietveld refinement as well as the crystallites’ size (based on XRD data).

Response:

Thank you very much for your suggestions. In the previous studies, we have tried to determine the particle size and element content of biosynthetic minerals (Song et al., 2018). The particle sizes of the samples were obtained by a laser particle size analyzer (Microtrac S3500, Microtrac Inc., Montgomeryville, U.S.A), the samples were ultrasound-assisted dispersed in water and then transported towards the measuring cell. The dried sample was placed in a measuring cup to measure the mineral elemental composition using an X-ray fluorescence spectrometer (XRF-1800, Shimadzu, Tokyo, Japan).

Unfortunately, we did not do this work in our study. We would like to do supplementary experiments in the laboratory. However, as we all know, influenced by COVID-19, our school laboratory has not been officially opened. Hope the reviewer can understand.

Song, Y.W.; Liu, Y.L.; Wang, H.R. Comparison of the biological and chemical synthesis of schwertmannite at a consistent Fe²⁺ oxidation efficiency and the effect of extracellular polymeric substances of Acidithiobacillus ferrooxidans on biomineralization. Materials, 2018, 11(9): DOI: 10.3390/ma11091739.

 

Comment 6: There is no discussion part in the manuscript. It should be added or section Results should be extended on the discussion of the results with literature data.

Response:

Thank you for your suggestions. In the revised manuscript, we have combined the results with the discussion, and supplemented or improved the discussion according to the reviewer's suggestions. Please refer to the revised manuscript (marked in red). Thank you again for your careful review.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript ‘The important role of dissolved oxygen supply regulated by hydraulic shear force in iron  hydroxysulfate minerals biosynthesis  by Yang et al., aimed at investigating biomineralization processes and influence of O2 concentration regulated by shear force.

The manuscript may help us extend our knowledge about Fe biomineralization processes under acidic conditions after careful editing and presenting the data in more clear way. This work is interesting and I think will appeal to a relatively broad audience. I do, however, have a few items that I think would enhance the impact of this work if addressed. The English grammar and word choices can be somewhat confusing in many locations and strong editing of this work would enhance its readability. Again, the underlying science appears sound but improving the flow of the writing will improve the overall caliber of the work.

 

Overall, the work is not clearly described and suffer from poor organization. I suggest the authors re structure and re-write the entire manuscript. Introduction section requires more clear explanation why there is a need such a study and what we miss in the current literature and suggest the authors read the recent papers to evaluate AMD formation in wide world and role of Fe chemical vs biological oxidation.  

 Method section needs more clarification in terms of describing key part of applied methods. For example, how the authors calculated TFe efficiency  is not clear. More importantly, adding of FeSO4 into the medium without sterilization may arise serious concern about the biological experimental set up. The authors need to justify this method that there was no contamination in the biological experiments.  The data used for figures should be provided in a table even as supplementary data.

 

Result  section deserve more thinking to present the data in more proper way and proper subtitles . In the current version , it is pretty difficult to follow and make sense things.

In the current version there is no discussion section which is important part of a scientific study to reveal significance of study as well as how these new data provide or extend or not extend our current understanding. I strongly urge the authors to add discussion part into the MS and discuss what these data means in terms of preventing AMD formation and/or developing rehabilitation strategies.

Please find the annotated MS for further explanation.

 

Comments for author File: Comments.pdf

Author Response

Point-by-point responses to the reviewers’ comments

Ms. Ref. No.: minerals-782178
Title: The important role of dissolved oxygen supply regulated by hydraulic shear force in the biosynthesis of iron hydroxysulfate minerals

Authors: Jun Yang, Rui Wang, Heru Wang, Zhenyu Wang and Yongwei Song *

 

Reviewer #2:

Comment 1: Overall, the work is not clearly described and suffer from poor organization. I suggest the authors re structure and re-write the entire manuscript. Introduction section requires more clear explanation why there is a need such a study and what we miss in the current literature and suggest the authors read the recent papers to evaluate AMD formation in wide world and role of Fe chemical vs biological oxidation.  

Response:

Thank you for pointing out the key issues, which is very helpful in improving the overall framework of the article. According to the reviewer's suggestions, and in order to improve the readability of the text, we have adjusted the structure of the text, supplemented the discussion section of the research results. Please refer to the revised manuscript (marked in red).

 

Comment 2: Method section needs more clarification in terms of describing key part of applied methods. For example, how the authors calculated TFe efficiency  is not clear. More importantly, adding of FeSO₄ into the medium without sterilization may arise serious concern about the biological experimental set up. The authors need to justify this method that there was no contamination in the biological experiments.  The data used for figures should be provided in a table even as supplementary data.

Response:

Thank you very much for your suggestion. We have revised the materials and methods section. For example, the Fe²⁺ oxidation efficiency and TFe precipitation efficiency were calculated according to the following formulas:

Fe²⁺ oxidation efficiency (%) = [(C_Fe(II) − C’_Fe(II))/C_Fe(II)] × 100%, where C_Fe(II) is the initial Fe²⁺ concentration (mg/L), and C’_Fe(II) is the Fe²⁺ concentration (mg/L) at different times.

TFe precipitation efficiency (%) = [(C_TFe − C’_TFe)/C_TFe] × 100%, where C_TFe is the initial TFe concentration (mg/L), and C’_TFe is the TFe concentration (mg/L) at different times.

In fact, during the experiment, FeSO₄ has been sterilized at high temperature. We are very sorry, this is a low-level error due to our negligence. In order to avoid misleading the readers, we have described it in the revised manuscript.

 

Comment 3: Result  section deserve more thinking to present the data in more proper way and proper subtitles . In the current version, it is pretty difficult to follow and make sense things.

Response:

Thank you very much for your suggestion. After careful consideration of your comments, we have made major changes to the result section. Please refer to the revised manuscript (marked in red).

 

Comment 4: In the current version there is no discussion section which is important part of a scientific study to reveal significance of study as well as how these new data provide or extend or not extend our current understanding. I strongly urge the authors to add discussion part into the MS and discuss what these data means in terms of preventing AMD formation and/or developing rehabilitation strategies.

Response:

Thank you for your suggestions. In the revised manuscript, we have combined the results with the discussion, and supplemented or improved the discussion according to the reviewer's suggestions. Please refer to the revised manuscript (marked in red). Thank you again for your careful review.

 

Comment 5: Please find the annotated MS for further explanation.

Response:

Thank you for your important comments. We appreciate the reviewer’s rigorous attitude. We have revised the manuscript according to your suggestions. Thank you very much for your help!

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors showed that the oxygen requirement at the intimal stage of Fe²⁺ bio-oxidation by A. ferrooxidans is important for overall precipitation of iron as hydroxysulfate minerals. Although the experiment was conducted nicely, the results are poorly discussed and needs improvement. Following are comments.

P 1, L 14. Mention the full form of A. ferrooxidans whenever used for the first time.

P 2, L 56-57. “In an extremely acidic environment, …….hydrolysis mineralization.” Revise the sentence.

P 2, L 57-59. Can you discuss more about the optimum growth of A. ferrooxidans at low pH range?

P 2, L 59. “It also uses CO2 as a carbon source to absorb N, P,…”. How assimilating CO2 would aid in absorbing other nutrients?

P 2, L 74-75. “9K medium-FeSO4-A. ferrooxidans solution”. It is not clear whether you developed or modified it? If the composition is taken from another study, then cite it. Write the name of the media correctly.

P 2, L 82-84. Revise the sentences.

P 2, L 84. Sterilized using what?

P 2, L 89. “…. inoculated with A. ferrooxidans to create a 10% (V/V) proportion”. The sentence is not clear, revise it.

P 2, L 90. “180 r/min”. is it RPM?

P 2, L 92. Provide the details of filter paper. Material, pore size, manufacture etc.

P 3, L 93. Washed with what?

P 3, L 95, L 103. What was the concentration (% v/v or mM) of H₂SO₄ solution?

P 3, L 96. Can you elaborate the double-layer plate method briefly?

P 3, L 93, L 104. “at 10000×g” and “240 r/min”. Can the units be used uniformly?

P 4, L 161. Where is the discussion part?

P 5, L 162. Why Subsection 3.1 heading is the effect of hydraulic sheer force when you are not directly showing the variation of sheer force rather discussing it indirectly through varying the shaking speed?  

P 5, L 181. Does 120–240 r/min indicate Fe3+ supply rates?

P 5, L 184-187. What about disruption of bacterial cells at high sheer force resulting from shaking at 240 r/min? Cite from other studies if there is a report of cell disruption at high sheer force.

P 5, L 188-189. Fig. 4 shows that the flask at 240 r/min had highest amount of DO all over the incubation period. Even if the bacteria can not adhere to the solid surface, is the amount of DO not enough to oxidize Fe²⁺ to Fe³⁺?

P 6, L 204. Why the DO in 0 r/min is higher than that of 60 r/min?

P 6, L 217. If the O₂ dissolution rate into the solution is low at higher sheer speed, then how it is showing high DO? If the O₂ is not enough for A. ferrooxidans mediated oxidation process at higher sheer speed, then how the Fe²⁺ decreased with increasing sheer speed to 120 and 180 r/min?

P 6, L 217-219. The meaning is not clear, revise the sentences.

P 7, L 256-259. How about the consumption of DO by A. ferrooxidans during the initial hours of incubation?

P 9, L 289-299. Why the low Fe²⁺ oxidation leads to the formation of schwertmannite, while the high Fe²⁺ oxidation leads to jarosite formation?

Author Response

Point-by-point responses to the reviewers’ comments

Ms. Ref. No.: minerals-782178
Title: The important role of dissolved oxygen supply regulated by hydraulic shear force in the biosynthesis of iron hydroxysulfate minerals

Authors: Jun Yang, Rui Wang, Heru Wang, Zhenyu Wang and Yongwei Song *

 

Reviewer #3:

The authors showed that the oxygen requirement at the intimal stage of Fe²⁺ bio-oxidation by A. ferrooxidans is important for overall precipitation of iron as hydroxysulfate minerals. Although the experiment was conducted nicely, the results are poorly discussed and needs improvement. Following are comments.

Comment 1: P 1, L 14. Mention the full form of A. ferrooxidans whenever used for the first time.

Response:

Thank you for your careful attitude. According to your suggestion, we have revised the full form of A. ferrooxidans.

 

Comment 2: P 2, L 56-57. “In an extremely acidic environment, …….hydrolysis mineralization.” Revise the sentence.

Response:

Thank you very much for pointing this out. According to your suggestions, we have revised the sentence to: In an extremely acidic environment, the A. ferrooxidans-mediated biomineralization involves biological oxidation of Fe²⁺ and Fe³⁺ hydrolysis reactions.

 

Comment 3: P 2, L 57-59. Can you discuss more about the optimum growth of A. ferrooxidans at low pH range?

Response:

Thank you for your comments. Previous studies showed that schwertmannite formation usually occurs in Fe and sulfate-rich solutions in a pH range of 2.5–4.5, with lower pH values promoting jarosite precipitation. For instance, Bigham et al. (1996) demonstrated that schwertmannite, formed through the bio-oxidation of Fe²⁺ by A. ferrooxidans, was the only mineral phase at pH=3.0. Liao et al. (2009) observed that the formed ferric hydroxysulfate minerals were a mixture of Na-jarosite and schwertmannite at an initial pH of 3.5, while pH in the range of 1.6–3.4 produced only pure schwertmannite.

Bigham, J.M.; Schwertmann, U.; Pfab, G. Influence of pH on mineral speciation in a bioreactor simulating acid mine drainage. Appl. Geochem. 1996, 11, 845–849.

 

Liao, Y.H.; Zhou, L.X.; Liang, J.R.; Xiong, H.X. Biosynthesis of schwertmannite by Acidithiobacillus ferrooxidans cell suspensions under different pH condition. Mat. Sci. Eng. C 2009, 29, 211–215.

 

Comment 4: P 2, L 59. “It also uses CO₂ as a carbon source to absorb N, P,…”. How assimilating CO₂ would aid in absorbing other nutrients?

Response:

Thank you very much for your reminding. Your opinion makes us realize that the expression of the sentence is wrong. We have revised the sentence to: It also uses CO₂ as a carbon source, N and P as nutrient elements, and then employs the energy from the Fe²⁺ oxidation to support its growth and reproduction.

 

Comment 5: P 2, L 74-75. “9K medium-FeSO₄-A. ferrooxidans solution”. It is not clear whether you developed or modified it? If the composition is taken from another study, then cite it. Write the name of the media correctly.

Response:

Thank you for pointing this out. Many literatures use this form to express the properties of media. Take the following literatures as example:

Bai, S.Y.; Xu, Z.H.; Wang, M.; Liao, Y.H.; Liang, J.R.; Zheng, C.C.; Zhou, L.X. Both initial concentrations of Fe(II) and monovalent cations jointly determine the formation of biogenic iron hydroxysulfate precipitates in acidic sulfate-rich environments. Materials Science and Engineering C 2012, 32, 2323–2329.

Bai, S.Y.; Liang, J.R.; Wang, M.; Zhou, L.X. Effect of Fe( III) supply rate on the formation of amorphous schwertmannite. Acta Miner. Sinica 2011, 31, 256–262.

Bai, S.Y.; Liang, J.R.; Zhou, L.X. Effectsof iron/potassium molar ratio on mass of biogenic Fe(Ⅲ) hydroxysulfate precipitates in the FeSO₄-K₂SO₄-H₂O system and their environmental implications. Acta Scientiae Circumstantiae 2010, 30, 1601–1607.

Bai, S.Y.; Liang, J.R.; Zhou, L.X. Effects of monovalent cation and dissolved organic matter on the formation of biogenic secondary iron minerals in bioleaching system. Acta Miner. Sinica 2011, 31, 118–125.

Song, Y.W.; Liu, Y.L.; Wang, H.R. Comparison of the biological and chemical synthesis of schwertmannite at a consistent Fe²⁺ oxidation efficiency and the effect of extracellular polymeric substances of Acidithiobacillus ferrooxidans on biomineralization. Materials, 2018, 11(9): DOI: 10.3390/ma11091739.

 

Comment 6: P 2, L 82-84. Revise the sentences.

Response:

Thank you for your suggestions. We have revised the sentence to: The modified 9K liquid medium was prepared as follows: 3.5 g of (NH₄)₂SO₄, 0.119 of KCl, 0.058 of K₂HPO₄, 0.0168 of Ca(NO₃)₂·4H₂O and 0.583 g of MgSO₄·7H₂O in 1 L of distilled water, adjusted to pH=2.50 with 1:1 (v/v) H₂SO₄, followed by sterilization using a sterilizer for 30 min at 121 °C.

 

Comment 7: P 2, L 84. Sterilized using what?

Response:

Please refer to comment 6's response. Thank you!

 

Comment 8: P 2, L 89. “…. inoculated with A. ferrooxidans to create a 10% (V/V) proportion”. The sentence is not clear, revise it.

Response:

Thank you for pointing this out. We have revised the sentence to: In an Erlenmeyer flask containing 225 mL of modified 9K liquid medium, inoculated with 25 mL of A. ferrooxidans to create a 10% (v/v) proportion, and then FeSO₄·7H₂O (sterilized for 30 min at 121 °C) was added to produce a Fe²⁺ concentration of 9.67 g/L.

 

Comment 9: P 2, L 90. “180 r/min”. is it RPM?

Response:

Yes, it’s “rpm”. We have replaced r/min by rpm throughout the entire text.

 

Comment 10: P 2, L 92. Provide the details of filter paper. Material, pore size, manufacture etc.

Response:

Thank you for your suggestion. We have added the details of filter paper in the revised manuscript. Please refer to the revised manuscript (marked in red).

 

Comment 11: P 3, L 93. Washed with what?

Response:

I'm sorry we didn't describe it clearly, here, the bacteria was washed twice with dilute H₂SO₄ (pH=1.50) to remove different doped ions.

 

Comment 12: P 3, L 95, L 103. What was the concentration (% v/v or mM) of H₂SO₄ solution?

Response:

The concentration of H₂SO₄ solution is 1:1 (v/v).

 

Comment 13: P 3, L 96. Can you elaborate the double-layer plate method briefly?

Response:

The high concentration of dissolved organic matter, which is toxic to chemoautotrophic Acidithiobacilli bacteria, released from agar with autoclave were responsible for the lower colony count efficiency for single-layer plate. On contrast, the yeast strain lived in underlayer consumed a lot of dissolved organic matter in medium, resulting in a higher colony count efficiency for double-layer plate. A double-layer plate approach was developed to increase the colony count efficiency of chemoautotrophic Acidithiobacilli bacteria. 0.1 mL of heterotrophic yeast strain Rhodotorula sp. R30 solution was spread onto the bottom layer made of 2% of agar in plate, and then, the mineral-salt medium containing 0.4% agarose for Acidithiobacillus ferrooxidans were poured on it as upper layer. After the plates were cold, 0.1 mL of the bacterial suspension containing Acidithiobacillus ferrooxidans was spread evenly on the superstratum. The double-layer plate was cultured at 30 ℃ (Wang et al. 2005).

Wang, S.M.; Zhou, L.X. A renovated approach for increasing colony count efficiency of Thiobacillus ferrooxidans and Thiobacillus thiooxidans: Double-layer plates. Acta Sci. Circum. 2005, 25, 1418–1420.

 

Comment 14: P 3, L 93, L 104. “at 10000×g” and “240 r/min”. Can the units be used uniformly?

Response:

Thank you for pointing this out. In order to unify the units, we have changed 10000×g to 7720 rpm in the revised manuscript.

 

Comment 15: P 4, L 161. Where is the discussion part?

Response:

Thank you for your suggestions. In the revised manuscript, we have combined the results with the discussion, and supplemented or improved the discussion according to the reviewer's suggestions. Please refer to the revised manuscript (marked in red). Thank you again for your careful review.

 

Comment 16: P 5, L 162. Why Subsection 3.1 heading is the effect of hydraulic sheer force when you are not directly showing the variation of sheer force rather discussing it indirectly through varying the shaking speed?  

Response:

Thank you for pointing this out. There are many ways to generate hydraulic shear force, such as agitation, oscillation or aeration. In this study, the hydraulic shear force achieved by regulating the shaking table’s rotation speed. Therefore, in the results and discussion section, the shaking speed is used to refer to the hydraulic shear force.

 

Comment 17: P 5, L 181. Does 120–240 r/min indicate Fe³⁺ supply rates?

Response:

Thank you very much for your reminding. Your opinion makes us realize that the sentence in the original manuscript is inaccurate. In a scientific and rigorous manner, and to avoid misleading the readers, we have deleted “(120–240 rpm)” of the sentence.

 

Comment 18: P 5, L 184-187. What about disruption of bacterial cells at high sheer force resulting from shaking at 240 r/min? Cite from other studies if there is a report of cell disruption at high sheer force.

Response:

Thank you for your suggestion. I'm very sorry that we can't find the relevant literature. However, according to the oxidation efficiency of Fe²⁺ in solution, Acidithiobacillus ferrooxidans has similar biological oxidation activity at 120–240 rpm. It also indicated that Acidithiobacillus ferrooxidans cells were not damaged in this speed range.

 

Comment 19: P 5, L 188-189. Fig. 4 shows that the flask at 240 r/min had highest amount of DO all over the incubation period. Even if the bacteria can not adhere to the solid surface, is the amount of DO not enough to oxidize Fe²⁺ to Fe³⁺?

Response:

It can be seen from Figure 1 that the oxidation efficiency of Fe²⁺ is basically the same in the speed range of 120–240 rpm, which indicates that the amount of DO in the solution is sufficient to support the biological oxidation process of Acidithiobacillus ferrooxidans. When the rotational speed of the shaker increases from 180 to 240 rpm, the strong collision of the solution on the inner walls of the Erlenmeyer flask produces abundant foam. I think this is the main reason why the flask at 240 rpm had highest amount of DO all over the incubation period.

 

Comment 20: P 6, L 204. Why the DO in 0 r/min is higher than that of 60 r/min?

Response:

When the shaking table’s rotation speed is 0 rpm, O₂ in the air is difficult to enter the solution, resulting in the gradual decrease of DO concentration in the solution during the cultivation process. However, due to the low utilization of DO by Acidithiobacillus ferrooxidans, more DO remained in the solution. When the rotating speed was 60 rpm, Acidithiobacillus ferrooxidans increased the utilization efficiency of DO, and the residual DO in the solution was low at the end of culture.

 

Comment 21: P 6, L 217. If the O₂ dissolution rate into the solution is low at higher sheer speed, then how it is showing high DO? If the O₂ is not enough for A. ferrooxidans mediated oxidation process at higher sheer speed, then how the Fe²⁺ decreased with increasing sheer speed to 120 and 180 r/min?

Response:

Thank you for your comments. Maybe because our statement is not accurate enough, the reviewer misunderstood the result of the text. In fact, the larger the hydraulic shear force is, the more beneficial it is to the dissolution of O₂, thus showing a high concentration of DO. In addition, under the condition of high hydraulic shear force (120–240 rpm), DO is enough to support the biological oxidation of Acidithiobacillus ferrooxidans. Please refer to comment 19's response. Thank you!

 

Comment 22: P 6, L 217-219. The meaning is not clear, revise the sentences.

Response:

Thank you for your suggestion. We have revised the sentence to: However, when the density of A. ferrooxidans is high, the concentration of Fe²⁺ will decrease gradually. At this time, the Fe²⁺ concentration becomes the main limiting factor of biological oxidation reaction.

 

Comment 23: P 7, L 256-259. How about the consumption of DO by A. ferrooxidans during the initial hours of incubation?

Response:

Unfortunately, we did not measure the consumption of DO by A. ferrooxidans. According to the results of other two treatments, the utilization ratio of DO by A. ferrooxidans was relatively low in 0–12h culture period, which may be caused by the fact that the cells had not yet adapted to the new environment.

 

Comment 24: P 9, L 289-299. Why the low Fe²⁺ oxidation leads to the formation of schwertmannite, while the high Fe²⁺ oxidation leads to jarosite formation?

Response:

Thank you for your comments. Bai et al. (2012) observed that either amorphous schwertmannite or crystalline jarosite existed at the critical point of jarosite formation in FeSO₄-K₂SO₄-H₂O system by A. ferrooxidans, depended on the rate of Fe³⁺ supply. The lower supply rate of Fe³⁺ obviously inhibited K⁺ incorporation into iron hydroxysulfate mineral to form jarosite, consequently improved the generation of amorphous schwertmannite in the system (Fig. 1). Meanwhile, the influence of initial Fe²⁺ concentration on the mineralogy of the precipitates formed in solutions containing 4 mmol/L of K⁺ was investigated. As shown in Fig. 2, the precipitate was the pure schwertmannite when the initial Fe²⁺ concentrations was below 40 mmol/L, while the secondary ferric minerals were mixtures of schwertmannite and jarosite when theinitial Fe²⁺ concentrations was above 80 mmol/L. In other words, at a fixed K⁺ concentration, lower initial Fe²⁺ concentration did not lead to the formation of jarosite. These results implied that the crystallinity of the secondary ferric minerals increased with the increase of initial Fe²⁺ concentration in the medium with a fixed K⁺ concentration. In order to make the readers better understand the reason, we explained it in the revised manuscript.

Fig. 1

Fig. 2

Bai, S.Y.; Xu, Z.H.; Wang, M.; Liao, Y.H.; Liang, J.R.; Zheng, C.C.; Zhou, L.X. Both initial concentrations of Fe(II) and monovalent cations jointly determine the formation of biogenic iron hydroxysulfate precipitates in acidic sulfate-rich environments. Mat. Sci. Eng. C 2012, 32, 2323–2329.

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Many thanks to the authors for their comprehensive responses to my comments. The paper is ready to be published in the journal.

Author Response

Point-by-point responses to the reviewers’ comments

Ms. Ref. No.: minerals-782178
Title: The important role of dissolved oxygen supply regulated by the hydraulic shear force during the biosynthesis of iron hydroxysulfate minerals

Authors: Jun Yang, Rui Wang, Heru Wang, Zhenyu Wang and Yongwei Song *

 

Reviewer #1:

Major concerns:

Comment: Many thanks to the authors for their comprehensive responses to my comments. The paper is ready to be published in the journal.

Response:

Thank you very much for your comments. With your help, the level of the revised manuscript has been greatly improved.

 

Reviewer #2:

Comment: The revised manuscript improve and many of concerns raised in my previous reviews have been covered. There are still a couple of minor issues need to be addressed before publications. Please find the annotated manuscript for further highlighted sections.

Response:

Thank you for your important comments. We appreciate the reviewer’s rigorous attitude. We have revised the manuscript according to your suggestions. Thank you very much for your help!

 

Reviewer #3:

The authors should provide line and page numbers of the revised manuscript in response to the reviewers' comments file. A few explanations are not included in the text. Authors should improve english. Following are some of the comments. 

 

Comment 1: Response to comment 2: Revise the sentence.

Response:

Thank you very much for pointing this out. According to your suggestions, we have revised the sentence to: In an extremely acidic environment, the A. ferrooxidans-mediated biomineralization involves the biological oxidation of Fe²⁺ and thus facilitate Fe hydrolysis. (P 2, L 67-68)

 

Comment 2: Response comment 8: Incorrect sentence formation, revise it. Improve english.  

Response:

Thank you! According to your suggestions, we have revised the sentence to: Sterilized FeSO₄·7H₂O was added to an Erlenmeyer flask containing 225 mL of the modified 9K liquid medium, which was inoculated with 25 mL of A. ferrooxidans to obtain an Fe²⁺ concentration of 9.67 g/L. (P 3, L 109-111)

 

Comment 3: Response to comment 10: The filter material, pore size and product part number are still missing.

Response:

I'm sorry we didn't describe it clearly, We have added the details of filter paper (1004-055 Whatman, Grade 4: 20–25 μm) in the revised manuscript. (P 3, L 113-114)

 

Comment 4: Response to comment 13: Did you add it to the text?

Response:

Thank you for your comments! We have supplemented the double-layer plate method in Section 2.5. (P 5, L 181-186)

 

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

The revised manuscript improve and many of concerns raised in my previous reviews have been covered. There are still a couple of minor issues need to be addressed before publications. Please find the annotated manuscript for further highlighted sections.

Comments for author File: Comments.pdf

Author Response

Point-by-point responses to the reviewers’ comments

Ms. Ref. No.: minerals-782178
Title: The important role of dissolved oxygen supply regulated by the hydraulic shear force during the biosynthesis of iron hydroxysulfate minerals

Authors: Jun Yang, Rui Wang, Heru Wang, Zhenyu Wang and Yongwei Song *

 

Reviewer #1:

Major concerns:

Comment: Many thanks to the authors for their comprehensive responses to my comments. The paper is ready to be published in the journal.

Response:

Thank you very much for your comments. With your help, the level of the revised manuscript has been greatly improved.

 

Reviewer #2:

Comment: The revised manuscript improve and many of concerns raised in my previous reviews have been covered. There are still a couple of minor issues need to be addressed before publications. Please find the annotated manuscript for further highlighted sections.

Response:

Thank you for your important comments. We appreciate the reviewer’s rigorous attitude. We have revised the manuscript according to your suggestions. Thank you very much for your help!

 

Reviewer #3:

The authors should provide line and page numbers of the revised manuscript in response to the reviewers' comments file. A few explanations are not included in the text. Authors should improve english. Following are some of the comments. 

 

Comment 1: Response to comment 2: Revise the sentence.

Response:

Thank you very much for pointing this out. According to your suggestions, we have revised the sentence to: In an extremely acidic environment, the A. ferrooxidans-mediated biomineralization involves the biological oxidation of Fe²⁺ and thus facilitate Fe hydrolysis. (P 2, L 67-68)

 

Comment 2: Response comment 8: Incorrect sentence formation, revise it. Improve english.  

Response:

Thank you! According to your suggestions, we have revised the sentence to: Sterilized FeSO₄·7H₂O was added to an Erlenmeyer flask containing 225 mL of the modified 9K liquid medium, which was inoculated with 25 mL of A. ferrooxidans to obtain an Fe²⁺ concentration of 9.67 g/L. (P 3, L 109-111)

 

Comment 3: Response to comment 10: The filter material, pore size and product part number are still missing.

Response:

I'm sorry we didn't describe it clearly, We have added the details of filter paper (1004-055 Whatman, Grade 4: 20–25 μm) in the revised manuscript. (P 3, L 113-114)

 

Comment 4: Response to comment 13: Did you add it to the text?

Response:

Thank you for your comments! We have supplemented the double-layer plate method in Section 2.5. (P 5, L 181-186)

 

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors should provide line and page numbers of the revised manuscript in response to the reviewers' comments file. A few explanations are not included in the text. Authors should improve english. Following are some of the comments. 

Response to comment 2: Revise the sentence.

Response to comment 6: Write the sentence correctly. What is the sterilizer, mention the name. 

Response comment 8: Incorrect sentence formation, revise it. Improve english.  

Response to comment 10: The filter material, pore size and product part number are still missing.

Response to comment 13: Did you add it to the text?

 

 

Author Response

Point-by-point responses to the reviewers’ comments

Ms. Ref. No.: minerals-782178
Title: The important role of dissolved oxygen supply regulated by the hydraulic shear force during the biosynthesis of iron hydroxysulfate minerals

Authors: Jun Yang, Rui Wang, Heru Wang, Zhenyu Wang and Yongwei Song *

 

Reviewer #1:

Major concerns:

Comment: Many thanks to the authors for their comprehensive responses to my comments. The paper is ready to be published in the journal.

Response:

Thank you very much for your comments. With your help, the level of the revised manuscript has been greatly improved.

 

Reviewer #2:

Comment: The revised manuscript improve and many of concerns raised in my previous reviews have been covered. There are still a couple of minor issues need to be addressed before publications. Please find the annotated manuscript for further highlighted sections.

Response:

Thank you for your important comments. We appreciate the reviewer’s rigorous attitude. We have revised the manuscript according to your suggestions. Thank you very much for your help!

 

Reviewer #3:

The authors should provide line and page numbers of the revised manuscript in response to the reviewers' comments file. A few explanations are not included in the text. Authors should improve english. Following are some of the comments. 

 

Comment 1: Response to comment 2: Revise the sentence.

Response:

Thank you very much for pointing this out. According to your suggestions, we have revised the sentence to: In an extremely acidic environment, the A. ferrooxidans-mediated biomineralization involves the biological oxidation of Fe²⁺ and thus facilitate Fe hydrolysis. (P 2, L 67-68)

 

Comment 2: Response comment 8: Incorrect sentence formation, revise it. Improve english.  

Response:

Thank you! According to your suggestions, we have revised the sentence to: Sterilized FeSO₄·7H₂O was added to an Erlenmeyer flask containing 225 mL of the modified 9K liquid medium, which was inoculated with 25 mL of A. ferrooxidans to obtain an Fe²⁺ concentration of 9.67 g/L. (P 3, L 109-111)

 

Comment 3: Response to comment 10: The filter material, pore size and product part number are still missing.

Response:

I'm sorry we didn't describe it clearly, We have added the details of filter paper (1004-055 Whatman, Grade 4: 20–25 μm) in the revised manuscript. (P 3, L 113-114)

 

Comment 4: Response to comment 13: Did you add it to the text?

Response:

Thank you for your comments! We have supplemented the double-layer plate method in Section 2.5. (P 5, L 181-186)

 

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf