Study on the Effectiveness of Sulfate Reducing Bacteria to Remove Heavy Metals (Fe, Mn, Cu, Cr) in Acid Mine Drainage

Round 1

Reviewer 1 Report

Overall the study  entitled: "Study on the effectiveness of Sulfate Reducing Bacteria to re-move heavy metals (Fe, Mn, Cu, Cr) in acid mine drainage" has significant merit and has the potential to contribute to the treatment of AMD using more environmentally friendly methods. The study could be considered for publication after adressing the following comments:

1. The study should be edited professionally or by a native English language speaker. The current grammatical errors and English usage detracts from an otherwise good product.

2. Rephrase the first sentence of the abstract - it is currently unclear what the authors try to convey in the statement: "As for the environmental pollution problems such as Fe, Mn, Cu, Cr and other heavy metals produced by acid mine drainage (AMD)."

3. Line 12: It the AMD real or synthetic AMD?

4. Line 36 (graphical abstract): The reference "Solution containing Fe²⁺..." should rather read "AMD solution containing..."

5. Line 73: The word "repair" here does not make sense. The AMD is not repaired as it is not itself broken, rather the AMD is treated or remediated.

6. Line 123: It is unclear if the AMD is simulated or authentic (from a site). If it is from a site, where was it obtained? If ti was simulated then it should be clearly stated and the preparation method reported.

7. Line 127: This seems to indicate that it was real AMD spiked with the desired heavy metals. This should be explicitly clarified.

8. Line 149: AA can only measure elements, not ions (total Fe, Mn, Cu, Cr were measured). It is inaccurate to imply that the ionic species were measured (Fe²⁺, Mn²⁺, Cu²⁺, Cr⁶⁺). This would have required an ion chromatograph. 

9. The reference to the ionic species in Figures 1 and 2 (e.g. Fe²⁺, Mn²⁺, etc.) should be corrected as these were not directly measured - rather indicate total Fe/Mn/etc. 

 

Author Response

To reviewer 1:

Thank you very much for your opinions. Combining with your opinions, we have revised the whole article. Your opinions are of great help to the improvement of all aspects of our paper. We marked the revisions in the article with yellow. Thank you very much for your opinion again. Best wishes. The following is our reply to your opinions one by one.

1. The study should be edited professionally or by a native English language speaker. The current grammatical errors and English usage detracts from an otherwise good product.

Replies: Thank you for your advice, we have invited the English teacher of our school to modify the grammar mistakes in this paper. The modified part were marked in yellow.

 

2. Rephrase the first sentence of the abstract - it is currently unclear what the authors try to convey in the statement: "As for the environmental pollution problems such as Fe, Mn, Cu, Cr and other heavy metals produced by acid mine drainage (AMD)."

Replies: Thank you for your advice, we checked the sentence again and found the grammar questions. We have changed the first sentence into "Acid Mine Drainage (AMD) emissions contains a large amount of heavy metals such as Fe, Mn, Cu, Zn, and so on, which has caused serious environmental contamination issues. To explore the effective reduction potential of heavy metals in AMD by SRB, we used the primary mixed Sulfate Reducing Bacteria (SRB) enriched from mining area soil, in the method of batch experiments, and add mixed SRB into the AMD containing different concentrations of Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺."

 

3. Line 12: It the AMD real or synthetic AMD?

Replies: Thank you for your comment, we checked the whole paper again and found that we didn"t indicate the source of the AMD. We have corrected the error, and changed the "AMD" into synthetic AMD.The AMD was taken from mining area and we have analyzed the various opponents in AMD. Because of the mining area we took samples from is situated in Shanxi Province, while our school laboratory is situated in Liaoning Province. The distance is really far, with a higher cost of transporting AMD, it"s really inconvenient. Therefore, based on the data of AMD we measured, the author explored the removal efficient of different heavy metals by SRB based on artificial simulated AMD. This study is still in the experimental stage. Later we will make further study and apply these experimental results in mining area in Shanxi Province. Meanwhile, the author has modified the "Materials and methods" section, and added some specific explanations on artificial simulated AMD, the details are as follows:

Synthetic AMD: Taking the AMD from the mining area in Shanxi province to make on-site-survey. After measurement, we found that the pH of the AMD was 5±0.1, and there were 2.0±0.05 g/L SO₄^2-, 0.6±0.05 g/L Na⁺, 0.04±0.01 g/L Ca²⁺, 0.20±0.05 g/L Mg²⁺, 0.15±0.02 g/L K⁺，0.28±0.02 g/L Fe²⁺, 0.018±0.02 g/L Mn²⁺, 0.013±0.02 g/L Cu²⁺, 0.05±0.01 g/L Cr⁶⁺ in AMD. Combining with the measured AMD data and the components of the Starkey medium, we made artificial simulated AMD in laboratory. The pH value was adjusted by 1 mol/L HNO₃ and 1 mol/L NaOH to make the artificial simulated AMD pH=5. At the same time the artificial simulated AMD we made containing 2.0 g/L SO₄^2-, 0.58 g/L Na⁺, 0.04 g/L Ca²⁺, 0.20 g/L Mg²⁺, 0.17 g/L K⁺, and a small amount of Cl^-, HPO₄^2-, sodium lactate. To explore the removal efficiency of different heavy metals of Fe, Mn, Cu, Cr, we added different concentrations of Fe²⁺ into artificial simulated AMD to form AMD solution containing Fe²⁺ of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L. Adding different concentrations of Mn²⁺ into synthetic AMD to form 0 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Mn²⁺. Adding different concentrations of Cu²⁺ into synthetic AMD to form 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Cu²⁺. Adding different concentrations of Cr⁶⁺ into synthetic AMD to form 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L AMD solution containing Cr⁶⁺. FeSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O and K₂CrO₄ produced by Tianjin Zhiyuan Chemical Reagent Co., Ltd. were used to prepare the solutions containing Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺ above. The above drugs are Analytical Reagent. The above solution was prepared with deionized water, and the ion contents in the above solution were measured by the instrument to avoid the error caused by the calculation.

 

4. Line 36 (graphical abstract): The reference "Solution containing Fe2+..." should rather read "AMD solution containing..."

Replies: Thank you for your suggestion. We have accepted your suggestion and corrected it.

 

5. Line 73: The word "repair" here does not make sense. The AMD is not repaired as it is not itself broken, rather the AMD is treated or remediated.

Replies: Thank you for your suggestion. We have realized the mistaken use of the word after you pointed that. We have changed the wrong word "repair" into the word "treat".

 

6. Line 123: It is unclear if the AMD is simulated or authentic (from a site). If it is from a site, where was it obtained? If it was simulated then it should be clearly stated and the preparation method reported.

Replies: Thank you for your suggestion. The author has supplemented some details about the artificial simulated AMD. The details are as follows:

Synthetic AMD: Taking the AMD from the mining area in Shanxi province to make on-site-survey. After measurement, we found that the pH of the AMD was 5±0.1, and there were 2.0±0.05 g/L SO₄^2-, 0.6±0.05 g/L Na⁺, 0.04±0.01 g/L Ca²⁺, 0.20±0.05 g/L Mg²⁺, 0.15±0.02 g/L K⁺，0.28±0.02 g/L Fe²⁺, 0.018±0.02 g/L Mn²⁺, 0.013±0.02 g/L Cu²⁺, 0.05±0.01 g/L Cr⁶⁺ in AMD. Combining with the measured AMD data and the components of the Starkey medium, we made artificial simulated AMD in laboratory. The pH value was adjusted by 1 mol/L HNO₃ and 1 mol/L NaOH to make the artificial simulated AMD pH=5. At the same time the artificial simulated AMD we made containing 2.0 g/L SO₄^2-, 0.58 g/L Na⁺, 0.04 g/L Ca²⁺, 0.20 g/L Mg²⁺, 0.17 g/L K⁺, and a small amount of Cl^-, HPO₄^2-, sodium lactate. To explore the removal efficiency of different heavy metals of Fe, Mn, Cu, Cr, we added different concentrations of Fe²⁺ into artificial simulated AMD to form AMD solution containing Fe²⁺ of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L. Adding different concentrations of Mn²⁺ into synthetic AMD to form 0 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Mn²⁺. Adding different concentrations of Cu²⁺ into synthetic AMD to form 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Cu²⁺. Adding different concentrations of Cr⁶⁺ into synthetic AMD to form 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L AMD solution containing Cr⁶⁺. FeSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O and K₂CrO₄ produced by Tianjin Zhiyuan Chemical Reagent Co., Ltd. were used to prepare the solutions containing Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺ above. The above drugs are Analytical Reagent. The above solution was prepared with deionized water, and the ion contents in the above solution were measured by the instrument to avoid the error caused by the calculation.

 

7. Line 127: This seems to indicate that it was real AMD spiked with the desired heavy metals. This should be explicitly clarified.

Replies: Thank you for your suggestion. The author has already corrected it. Please look the replies to question3 and question6 for more details.

 

8. Line 149: AA can only measure elements, not ions (total Fe, Mn, Cu, Cr were measured). It is inaccurate to imply that the ionic species were measured (Fe2+, Mn2+, Cu2+, Cr6+). This would have required an ion chromatograph.

Replies：Thank you for your advice. We have accepted your opinion and modified the paper. We have changed the Fe²⁺, Mn²⁺, Cu²⁺, Cr⁶⁺，in the initial version into total Fe, total Mn, total Cu, total Cr. As for the primitive artificial simulated AMD, we used the FeSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O and K₂CrO₄ to configure it. So the descriptions on "initial solution concentrations" we still used descriptions of ionic species. The part we modified has been marked in yellow.

 

9. The reference to the ionic species in Figures 1 and 2 (e.g. Fe2+, Mn2+, etc.) should be corrected as these were not directly measured - rather indicate total Fe/Mn/etc.

Replies: Thank you for your suggestion. we have accepted your opinion, please see question 8 for details.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, SRB in removing heavy metals of Fe, Mn, Cu, Cr in simulated acid mine drainage was investigated. Some of the metals can be removed effectively, and the mechanism of Fe2+ removal is investigated. The SRB method is environment-friendly and needs to be further investigated. But, the experimental methods were not new and only simulated AMD was used. Besides, the English needs to be improved. The specific comments are as follows:

(1)   As sediments of the insoluble metal salts were produced during the treatments, which may increase the turbidity of the liquid. So, if the cell growth measured by OD600 could be interfered.

(2)   Line 123-125: “the AMD used for the test contained ….” changes to “The simulated AMD used for the test contained …”.

(3)   The SRB culture preservation and cultivation methods should be given in the Methods and Materials section.

(4)   It would be better to test the SRB using real AMD.

(5)   The English is recommended to be revised by a native English speaker.

The meaning of some expressions, like “… to make images” in line 139, is not correct.

There are many grammatical errors. For example, there are no verb in the following sentences:

The first sentence of Abstract: “As for the environmental pollution problems such as Fe, Mn, Cu, Cr and other heavy 10 metals produced by acid mine drainage (AMD).”

In line 122-123: ” Combining with the composition of AMD and bacterial media [24], to configure the AMD used for the test.”

Author Response

Response to reviewers

Dear Editor and Reviewers,

 

Thank you for offering us an opportunity to improve the quality of our submitted manuscript (sustainability-2254196). We appreciated very much the reviewers' constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. We hope the revised manuscript has now met the publication standard of your journal.

 

We highlighted all the revisions in yellow color.

 

On the next pages, our point-to-point responses to the queries raised by the reviewers are listed.

 

 

Yours sincerely,

Yanrong Dong

Email: dongyanrong@lntu.edu.cn 

Telephone: +86-18241891851

Address: College of Civil Engineering, Liaoning Technical University No.47 Zhonghua Road, Xihe District, Fuxin, Liaoning 123000, China

 

 

To reviewer 2:

(1)As sediments of the insoluble metal salts were produced during the treatments, which may increase the turbidity of the liquid. So, if the cell growth measured by OD600 could be interfered.

Replies:Thank you for your suggestion. All the samples we used have been filtrated by filter paper before detection, as well as the blank group. Thus avoiding the measurement errors of OD₆₀₀. Meanwhile, we have already modified the "Materials and methods" section, and added some details, which are shown as follows:

"The pH value, ORP value, OD₆₀₀ value and Ec value were measured by PHS-3C pH meter (Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China), CT-8022 ORP meter (Shanghai Hechen Energy Technology Co., Ltd. , Shanghai, China) and V-1600PC visible spectrophotometer (Shanghai Yuanwang Liquid Level Meter Co., Ltd., Shanghai, China). The samples to be tested were filtered with 0.22 μm filter membrane."

 

(2)Line 123-125: “the AMD used for the test contained ….” changes to “The simulated AMD used for the test contained …”.

Replies: Thank you for your suggestion. We accepted your suggestion and corrected it.

 

(3)The SRB culture preservation and cultivation methods should be given in the Methods and Materials section.

Replies: Thank you for your advice, we have added this part in "Methods and Materials" section.

And the details is shown as follows:

SRB: The SRB we used was the mixed SRB cultured in laboratory. Mixed SRB was enriched from the wet soil in Haizhou open-pit coal gangue mountain in Fuxin City, Liaoning Province (121°41′E, 41°59′N) [24]. Wet soil was inoculated into sterile Starkey medium for anaerobic culture, which components were 0.5 g K₂HPO₄, 1.0 g NH₄Cl, 2.0 g MgSO₄·7H₂O, 0.5 g Na₂SO₄, 0.1 g CaCl₂·H₂O, 1.0 g yeast extract, 4 mL sodium lactate, 0.5 g (NH₄)₂Fe(SO₄)₂·6H₂O, 0.1 g ascorbic acid, 1 L distilled water, sterilizing for 30 min at pH=7, 121℃. Among them, (NH₄)₂Fe(SO₄)₂ and ascorbic acid can"t be sterilized at high temperature, so we used 0.22 μm filter membrane to filtrate and sterilize. When producing H₂S and emerging a large amount of black precipitates in the medium, it indicated that the mixed SRB has been cultured [24]. SRB in logarithmic growth phase was used to remove heavy metal ions by SRB. There were three replicates for each test setting. SRB was preserved in the method of glycerol tube cryopreservation, which was in sterile operating conditions, adding 500 μL bacterial solution and 500 μL 50 % glycerol solution into the sterilized 2 mL cryopreservation tube, mixing the solution lightly and preserving the mixed solution at -80°C.

 

(4)It would be better to test the SRB using real AMD.

Replies: Thank you for your opinions. The AMD was taken from mining area and we have analyzed the various opponents in AMD. Because of the mining area we took samples from is situated in Shanxi Province, while our school laboratory is situated in Liaoning Province. The distance is really far, with a higher cost of transporting AMD, it"s really inconvenient. Therefore, based on the data of AMD we measured, the author explored the removal efficient of different heavy metals by SRB based on artificial simulated AMD. This study is still in the experimental stage. Later we will make further study and apply these experimental results in mining area in Shanxi Province. Meanwhile, the author has modified the "Materials and methods" section, and added some specific explanations on artificial simulated AMD, the details are as follows:

SRB: The SRB we used was the mixed SRB cultured in laboratory. Mixed SRB was enriched from the wet soil in Haizhou open-pit coal gangue mountain in Fuxin City, Liaoning Province (121°41′E, 41°59′N) [24]. Wet soil was inoculated into sterile Starkey medium for anaerobic culture, which components were 0.5 g K₂HPO₄, 1.0 g NH₄Cl, 2.0 g MgSO₄·7H₂O, 0.5 g Na₂SO₄, 0.1 g CaCl₂·H₂O, 1.0 g yeast extract, 4 mL sodium lactate, 0.5 g (NH₄)₂Fe(SO₄)₂·6H₂O, 0.1 g ascorbic acid, 1 L distilled water, sterilizing for 30 min at pH=7, 121℃. Among them, (NH₄)₂Fe(SO₄)₂ and ascorbic acid can"t be sterilized at high temperature, so we used 0.22 μm filter membrane to filtrate and sterilize. When producing H₂S and emerging a large amount of black precipitates in the medium, it indicated that the mixed SRB has been cultured [24]. SRB in logarithmic growth phase was used to remove heavy metal ions by SRB. There were three replicates for each test setting. SRB was preserved in the method of glycerol tube cryopreservation, which was in sterile operating conditions, adding 500 μL bacterial solution and 500 μL 50 % glycerol solution into the sterilized 2 mL cryopreservation tube, mixing the solution lightly and preserving the mixed solution at -80°C.

 

(5)The English is recommended to be revised by a native English speaker.

The meaning of some expressions, like “… to make images” in line 139, is not correct.

There are many grammatical errors. For example, there are no verb in the following sentences:

The first sentence of Abstract: “As for the environmental pollution problems such as Fe, Mn, Cu, Cr and other heavy 10 metals produced by acid mine drainage (AMD).”

In line 122-123: ” Combining with the composition of AMD and bacterial media [24], to configure the AMD used for the test.”

Replies: Thank you for your suggestion. As for the grammar question you have mentioned, we have revised the language problems of this paper again. As for the sentence "line 139, the first sentence of the abstract, line 122-123" you took for examples, we have already corrected them either.

Among them, the first sentence was amended to " Aiming at the problem of environmental pollution caused by heavy metals such as Fe, Mn, Cu and Cr. Sulfate Reducing Bacteria (SRB) were enriched in mining soil. SRB was added to AMD containing different concentrations of Fe, Mn, Cu and Cr by batch experiments to explore the treatment potential of SRB on heavy metals in AMD.

The sentence of line 122-123 was amended to "Combining with the composition of AMD and bacterial media [24], we configured the AMD used for the test".

The sentence of line 139 was amended to "Taking the average value and errors of three repeated tests to figure."

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript is a case study on the effectiveness of sulfate reducing bacteria to remove heavy metals in acid mine drainage. The topic is of interest since the heavy metal pollution is a stringent environmental problem.

 

Some improvements are necessary to increase the manuscript quality, as follows:

1.      Please provide the e-mail addresses for each affiliation

2.      Abstract: the first sentence should be rewritten to be understood. Actually, the whole abstract should be rewritten to be self-explanatory for the whole manuscript

3.      Keywords: please remove the keywords already in the title and include pertinent keywords specific to the article not find in the title. “remove” is not an appropriate keyword

4.      Introduction: The text is very difficult to be read and understood, the whole content should be rephrased.

5.      Material and methods: please provide the name of equipment producers, city and country (e.g.  PHS-3C pH meter, 1600 PC visible spectrophotometer and Z-2000 flame atomic spectrophotometer).

6.      Material and methods: which substance was used to prepare Cr⁶⁺ solutions?

7.      Material and methods: please provide how it was assured the quality control for the analytical methods, how the analytical instruments were calibrated?

8.   Results and discussion: it is generally difficult to be read, please improve the language quality. The manuscript should be checked by a native English speaker.

Author Response

Response to reviewers

Dear Editor and Reviewers,

 

Thank you for offering us an opportunity to improve the quality of our submitted manuscript (sustainability-2254196). We appreciated very much the reviewers' constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. We hope the revised manuscript has now met the publication standard of your journal.

 

We highlighted all the revisions in yellow color.

 

On the next pages, our point-to-point responses to the queries raised by the reviewers are listed.

 

 

Yours sincerely,

Yanrong Dong

Email: dongyanrong@lntu.edu.cn 

Telephone: +86-18241891851

Address: College of Civil Engineering, Liaoning Technical University No.47 Zhonghua Road, Xihe District, Fuxin, Liaoning 123000, China

 

 

To reviewer 3:

1.Please provide the e-mail addresses for each affiliation

Replies: Thank you for your advice. We have already added e-mail address in the paper, the content we added had been marked in yellow.

 

2.Abstract: the first sentence should be rewritten to be understood. Actually, the whole abstract should be rewritten to be self-explanatory for the whole manuscript

Replies: Thank you for your advice, we have already reorganized the language and rewritten the abstract section. The details are shown as follows:

Aiming at the problem of environmental pollution caused by heavy metals such as Fe, Mn, Cu and Cr. Sulfate Reducing Bacteria (SRB) were enriched in mining soil. SRB was added to AMD containing different concentrations of Fe, Mn, Cu and Cr by batch experiments to explore the treatment potential of SRB on heavy metals in AMD. Combining with Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) to test, revealing the mechanism of removing heavy metal ions by SRB. The results showed that SRB had a strong ability to remove total Fe in the concentration range of 0-300 mg/L, and the removal percentage of total Fe reached to 92.16-93.17%. Besides, SRB had a good removal effect on total Cu in the concentration range of 0-20 mg/L, and the removal percentage of total Cu reached to 79.79-81.80%. SRB also had a good removal effect on total Cr, and the SRB activity probably would not be inhibited when the total Cr concentration was below 500 mg/L. The ability to solidify different heavy metal ions by SRB was: total Cr＞total Fe＞total Mn＞total Cu. When SRB removed Fe²⁺, there was precipitation shown as black granular at the same time. The precipitation was mainly FeS, containing a small amount of Fe₃(PO₄)₂·8H₂O, FeCO₃, Fe(OH)₂, Fe(OH)₃, Fe₂S as well.

 

3.Keywords: please remove the keywords already in the title and include pertinent keywords specific to the article not find in the title. “remove” is not an appropriate keyword

Replies: Thank you for your suggestion. We have already revised the keywords section, the modified keywords are: mining area, Sulfate Reducing Bacteria (SRB); heavy metal ions; removal mechanism.

 

4.Introduction: The text is very difficult to be read and understood, the whole content should be rephrased.

Replies: Thank you for your advice. We have already modified the introduction section. The modified part has been marked in yellow.

 

5.Material and methods: please provide the name of equipment producers, city and country (e.g.PHS-3C pH meter, 1600 PC visible spectrophotometer and Z-2000 flame atomic spectrophotometer).

Replies: Thank you for your suggestion, we have already added the lost information of the equipment into the paper in "Materials and methods" section which was marked in yellow. The details are shown as follows:

constant temperature oscillation incubator (Changzhou Kaihang Instrument Co., Ltd., Changzhou, China, type HZ-9811K);

PHS-3C pH meter (Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China);

CT-8022 ORP meter (Shanghai Hechen Energy Technology Co., Ltd. , Shanghai, China);

V-1600PC visible spectrophotometer (Shanghai Yuanwang Liquid Level Meter Co., Ltd., Shanghai, China);

Z-2000 flame atomic spectrophotometer (Hitachi society in Japan, Japan);

 

6.Material and methods: which substance was used to prepare Cr6+ solutions?

Replies:Thank you for your advice. The Cr⁶⁺ we used was configured by K₂CrO₄. And this part was added into the part of "artificial simulated AMD" in "Materials and methods" section. The details are shown as follows:

"FeSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O and K₂CrO₄ produced by Tianjin Zhiyuan Chemical Reagent Co., Ltd. were used to prepare the solutions containing Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺ above, The above drugs are Analytical Reagent. The above solution was prepared with deionized water."

 

7.Material and methods: please provide how it was assured the quality control for the analytical methods, how the analytical instruments were calibrated?

Replies: Thank you for your advice, In order to ensure the accuracy of the data, the ion contents we used are based on the data measured by the equipment. Besides, we took three replicates for each test setting, and made blank group to ensure the accuracy of our data. The conditions of the blank group were the same as the experimental group except SRB addition. And both of them have been taken the same operation steps to analyze the water quality indexes quantitively. Besides, we took repeated trial methods, which was the same experimental group had been repeated for 3 times to avoid the occasionality of the data. Meanwhile, the author added some details on "Materials and method" section, which are shown as follows:

SRB: The SRB we used was the mixed SRB cultured in laboratory. Mixed SRB was enriched from the wet soil in Haizhou open-pit coal gangue mountain in Fuxin City, Liaoning Province (121°41′E, 41°59′N) [24]. Wet soil was inoculated into sterile Starkey medium for anaerobic culture, which components were 0.5 g K₂HPO₄, 1.0 g NH₄Cl, 2.0 g MgSO₄·7H₂O, 0.5 g Na₂SO₄, 0.1 g CaCl₂·H₂O, 1.0 g yeast extract, 4 mL sodium lactate, 0.5 g (NH₄)₂Fe(SO₄)₂·6H₂O, 0.1 g ascorbic acid, 1 L distilled water, sterilizing for 30 min at pH=7, 121℃. Among them, (NH₄)₂Fe(SO₄)₂ and ascorbic acid can"t be sterilized at high temperature, so we used 0.22 μm filter membrane to filtrate and sterilize. When producing H₂S and emerging a large amount of black precipitates in the medium, it indicated that the mixed SRB has been cultured [24]. SRB in logarithmic growth phase was used to remove heavy metal ions by SRB. There were three replicates for each test setting. SRB was preserved in the method of glycerol tube cryopreservation, which was in sterile operating conditions, adding 500 μL bacterial solution and 500 μL 50 % glycerol solution into the sterilized 2 mL cryopreservation tube, mixing the solution lightly and preserving the mixed solution at -80°C.

Synthetic AMD: Taking the AMD from the mining area in Shanxi province to make on-site-survey. After measurement, we found that the pH of the AMD was 5±0.1, and there were 2.0±0.05 g/L SO₄^2-, 0.6±0.05 g/L Na⁺, 0.04±0.01 g/L Ca²⁺, 0.20±0.05 g/L Mg²⁺, 0.15±0.02 g/L K⁺，0.28±0.02 g/L Fe²⁺, 0.018±0.02 g/L Mn²⁺, 0.013±0.02 g/L Cu²⁺, 0.05±0.01 g/L Cr⁶⁺ in AMD. Combining with the measured AMD data and the components of the Starkey medium, we made artificial simulated AMD in laboratory. The pH value was adjusted by 1 mol/L HNO₃ and 1 mol/L NaOH to make the artificial simulated AMD pH=5. At the same time the artificial simulated AMD we made containing 2.0 g/L SO₄^2-, 0.58 g/L Na⁺, 0.04 g/L Ca²⁺, 0.20 g/L Mg²⁺, 0.17 g/L K⁺, and a small amount of Cl^-, HPO₄^2-, sodium lactate. To explore the removal efficiency of different heavy metals of Fe, Mn, Cu, Cr, we added different concentrations of Fe²⁺ into artificial simulated AMD to form AMD solution containing Fe²⁺ of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L. Adding different concentrations of Mn²⁺ into synthetic AMD to form 0 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Mn²⁺. Adding different concentrations of Cu²⁺ into synthetic AMD to form 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Cu²⁺. Adding different concentrations of Cr⁶⁺ into synthetic AMD to form 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L AMD solution containing Cr⁶⁺. FeSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O and K₂CrO₄ produced by Tianjin Zhiyuan Chemical Reagent Co., Ltd. were used to prepare the solutions containing Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺ above. The above drugs are Analytical Reagent. The above solution was prepared with deionized water, and the ion contents in the above solution were measured by the instrument to avoid the error caused by the calculation.

Batch experiments were carried out to remove total Fe, total Mn, total Cu and total Cr by SRB. Among them, the test method of Fe removal by SRB: SRB at logarithmic growth stage was inoculated at 5% inoculum into AMD solutions containing Fe ion concentrations of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L. Then 10 mL of sterilized liquid paraffin was added to the above mixture and the conical flask was sealed with a rubber stopper. The conical flask was placed in a constant temperature oscillation incubator (Changzhou Kaihang Instrument Co., Ltd., Changzhou, China, type HZ-9811K) at 35°C and 150 r/min for oscillation culture. There were three replicates for each test setting. Samples were taken at 1 d intervals. Using sterilised AMD without SRB inoculation as a blank group.The pH value, ORP value, OD₆₀₀ value and Ec value were measured by PHS-3C pH meter (Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China), CT-8022 ORP meter (Shanghai Hechen Energy Technology Co., Ltd. , Shanghai, China) and V-1600PC visible spectrophotometer (Shanghai Yuanwang Liquid Level Meter Co., Ltd., Shanghai, China). The samples to be tested were filtered with 0.22 μm filter membrane. The concentrations of SO₄^2- and total Fe in the filtered samples were measured by V-1600 PC visible spectrophotometer and Z-2000 flame atomic spectrophotometer (Hitachi society in Japan, Japan), and the removal percentage of SO₄^2- and total Fe were calculated. The average value and error of the three groups of repeated tests were used to draw the image. The experimental method of removing total Mn, Cu, Cr by SRB was consistent with that of removing total Fe by SRB.

 

8.Results and discussion: it is generally difficult to be read, please improve the language quality. The manuscript should be checked by a native English speaker.

Replies:

Thank you for your suggestion, we have invited the English teacher of our school to check the grammar mistakes of the whole paper.

 

 

Author Response File: Author Response.pdf

Reviewer 4 Report

The abstract needs to be fully rewritten, it doesn't have a proper structure (introduction, materials and methods, results, discussion, and conclusions). The first sentence doesn't make any sense.

Lines 30, 48, 50, adjectives are not correctly used (seriously, acidity, seriously).

Line 51, worldwide misuse.

Line 59, attracted, not atrracted.

Line 66, have, not has.

Lin 70-72, repetitive nomenclature. 

Line 73, repair?

Line 87, were, not was. Check the writing in lines 88 and 89.

Line 108-115, this paragraph doesn't follow any proper way to end the introduction, it already concludes the research without covering materials, methods, and results. The introduction should end with the objective of the research, not some attempt to enumerate the performed activities.

Line 131, ..

Line 164, 173, 175, 176, 265, 323, were, not was.

Line 188, was more... what?

Line 190, was less...?

In general, some parts of the results and discussion section are just textual descriptions of the Figures, which is repetitive and not very insightful. It tends to be tedious and lacks more profound analyses. Further discussion should be included instead of describing what we can already see in the Figures.

Lines 331-332, "-355 mV, -351 mV, -235 mV, -153 mV, -131 mV and 3.14 mS/cm, 3.24 mS/cm, 3.77 331 mS/cm, 4.14 mS/cm, 4.34 mS/cm"? What is the meaning of the negative signs? Is there a correlation with reduction or oxidation with the ORP signs?

No statistical analyses were performed or otherwise indicated. To conclude without them is just wrong for so many reasons.

Figure 5 should be rearranged to have a better view of the details.

The conclusions are a summary of the results, not conclusions per se.

Finally, the blank group was mentioned but it was never shown.

Author Response

Response to reviewers

Dear Editor and Reviewers,

 

Thank you for offering us an opportunity to improve the quality of our submitted manuscript (sustainability-2254196). We appreciated very much the reviewers' constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. We hope the revised manuscript has now met the publication standard of your journal.

 

We highlighted all the revisions in yellow color.

 

On the next pages, our point-to-point responses to the queries raised by the reviewers are listed.

 

 

Yours sincerely,

Yanrong Dong

Email: dongyanrong@lntu.edu.cn 

Telephone: +86-18241891851

Address: College of Civil Engineering, Liaoning Technical University No.47 Zhonghua Road, Xihe District, Fuxin, Liaoning 123000, China

 

 

To reviewer 4:

Lines 30, 48, 50, adjectives are not correctly used (seriously, acidity, seriously).

Replies: Thank you for your suggestion, we have corrected the mistake. We have changed the wrong words in line 30 "seriously", the wrong words in line 48 "acidity", and the wrong word in line 50 "seriously", into the word "serious", "acidic", and "serious", respectively

 

Line 51, worldwide misuse.

Replies: Thank you for your advice. We have corrected the wrong words, the modified content are shown as follows:

Heavy metal pollution has become a global problem to be solved.

 

Line 59, attracted, not atrracted.

Replies: Thank you for your advice, we have been corrected the spelling mistake

 

Line 66, have, not has.

Replies: Thank you for your advice, we have been corrected the grammar mistake. Besides, we have also checked the whole paper, and corrected the grammar mistakes and the misuse of the words.

 

Lin 70-72, repetitive nomenclature. 

Replies: Thank you for your advice. Combining with you advice, we have rewritten the whole passage, and the modified version is shown as follows:

Therefore, based on the metabolic characteristics of SRB, SRB has been widely used in the field of AMD treatment. SRB can efficiently remove heavy metal ions and SO₄^2- in AMD [14], and reduce the environmental pollution caused by AMD.

 

Line 73, repair?

Replies: Thank you for your advice, we have changed the improper word into treat.

 

Line 87, were, not was. Check the writing in lines 88 and 89.

Replies: Thank you for your advice, we have corrected the grammar mistake.

 

Line 108-115, this paragraph doesn't follow any proper way to end the introduction, it already concludes the research without covering materials, methods, and results. The introduction should end with the objective of the research, not some attempt to enumerate the performed activities.

Replies: Thank you for your advice, we have rewritten the end of the introduction, the modified version is shown as follows:

Therefore, we got the primary mixed SRB enriched from the mining area soil samples. In the method of serial experiments, inoculating SRB into AMD containing different concentrations of Fe²⁺, Mn²⁺, Cu²⁺, Cr⁶⁺. Combining with the changes in the value of OD600, pH, oxidation reduction potential (ORP), electrical conductance (Ec) and the removal efficiency of SO₄^2-, total Fe, total Mn, total Cu, total Cr, we analyzed the effective reduction potential of heavy metals and sulfate in AMD by SRB. Combining with SEM-EDS, XRD and XPS test, we explored the mechanism of removing heavy metals by SRB.

 

Line 131, ..

Replies: Thank you for your advice, we have corrected the format errors.

 

Line 164, 173, 175, 176, 265, 323, were, not was.

Replies: Thank you for your advice, we have corrected the grammar mistake.

 

Line 188, was more... what?

Replies: Thank you for your advice, we have modified the grammar mistake, the modified sentence is shown as follows:

In the early stage, SRB metabolism was high and had stronger dissimilatory SO₄^2- reduction effect, and the removal percentage of SO₄^2- increased rapidly.

 

Line 190, was less...?

Replies: Thank you for your advice, we have modified the grammar mistake, the modified sentence is shown as follows:

In the later stage, SRB gradually entered the decline period, and the metabolism was slow, with less SO₄^2- being reduced, and the removal rate of SO₄^2- tended to be stable.

 

Question: In general, some parts of the results and discussion section are just textual descriptions of the Figures, which is repetitive and not very insightful. It tends to be tedious and lacks more profound analyses. Further discussion should be included instead of describing what we can already see in the Figures.

Replies: Thank you for your advice, we have modified the "results and discussion" section, deleting some repetitive sentences, adding some analysis content. The modified version is marked in yellow in paper.

 

Question: Lines 331-332, "-355 mV, -351 mV, -235 mV, -153 mV, -131 mV and 3.14 mS/cm, 3.24 mS/cm, 3.77 331 mS/cm, 4.14 mS/cm, 4.34 mS/cm"? What is the meaning of the negative signs? Is there a correlation with reduction or oxidation with the ORP signs?

Replies: ORP value was detected by CT-8022 ORP meter (Shanghai Hechen Energy Technology Co., Ltd. , Shanghai, China), negative signs indicated that the solution had certain reducibility. Combined with the SRB metabolic sulfate equation, it can also be seen when the ORP is negative, indicating that the SRB metabolic sulfate activity is strong.

AMP^4-+SO₄^2-+H⁺→APS^2-+HP₂O₇^3-                        (1)

APS^2-+2e^2-+H⁺→HSO₃^-+AMP^2-    E₀=-60 mV              (2)

HSO₃^-+6e^2-+6H⁺→HS^-+H₂O      E₀=-116 mV             (3)

 

Question: No statistical analyses were performed or otherwise indicated. To conclude without them is just wrong for so many reasons.

Replies:

Combining with you opinions, we added some analysis of relevant values and SRB.

 

Question: Figure 5 should be rearranged to have a better view of the details.

Replies: Thank you for your advice, we have changed the figure 5 into more clear images.

 

Question: The conclusions are a summary of the results, not conclusions per se.

Replies: Thank you for your advice, we have corrected the conclusion section, which is shown as follows:

Aiming at the problem of environmental pollution caused by heavy metals such as Fe, Mn, Cu and Cr. The enrichment method was used to obtain the primary mixed SRB from the soil samples of the mining area. Based on batch experiments, SRB was inoculated into AMD containing different concentrations of Fe^2+, Mn²⁺, Cu²⁺ and Cr⁶⁺. Combined with the changes of OD₆₀₀, pH, ORP and Ec values and the removal effects of SO₄^2-, Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺, the effective reduction potential of SRB on sulfate and heavy metals in acid mine drainage was analyzed. SRB had a strong ability to remove Fe ion among 0-300 mg/L, and the removal percentage of Fe ion reached 92.16-93.17%. When the initial Fe ion concentration weas 300 mg/L, the growth state of SRB was the best. The maximum values of OD₆₀₀, pH value, SO₄^2- removal percentage and Fe²⁺ removal percentage was 1.27, 6.91, 88.05% and 93.17%, respectively. The minimum values of ORP and Ec were -399 mV and 2.72 mS/cm, respectively. SRB had a strong ability to remove Mn²⁺ among 0-40 mg/L and the removal percentage was 79-86%. When the concentration of Mn²⁺ was 40 mg/L, the growth state of SRB was the best, and the maximum removal percentages of SO₄^2- and Mn²⁺ were 84.98% and 79.06%, respectively. SRB had a good removal effect on Cu²⁺ among 0-20 mg/L, and the removal percentage of Cu²⁺ was 79.79-81.80%. When Cu²⁺ exceeded 20 mg/L, the activity of SRB was inhibited. When the concentration of Cu²⁺ was 20 mg/L, the maximum removal percentages of SO₄^2- and Cu²⁺ were 78.78% and 79.79%, respectively. SRB had a good removal effect on Cr ion, and when Cr ion concentration was below 500 mg/L, it would not inhibit the activity of SRB. The removal percentage of SO₄^2- and Cr ion among 100-500 mg/L Cr ion by SRB were stable at 71.06-78.37% and 83.51-87.21%, respectively. SRB can metabolize SO₄^2- to form heavy metal sulfide-based precipitation. The immobilization ability of SRB to different heavy metal ions was: Cr⁶⁺>Fe²⁺>Mn²⁺>Cu²⁺. Combined with SEM-EDS, XRD and XPS tests, the mechanism of SRB removing heavy metal ions was explored. It was found that the precipitates formed when SRB removed Fe²⁺ were black granular. The precipitate was mainly FeS, and contains a small amount of Fe₃(PO₄)₂·8H₂O, FeCO₃, Fe(OH)₂, Fe(OH)₃, Fe₂S, etc.

 

Question: Finally, the blank group was mentioned but it was never shown.

Replies: Thank you for your opinion, we described the blank control group in the experimental materials and methods section. When using the instrument to detect, we use the blank experiment as the benchmark for detection, and the experimental results are obtained by the difference of the data displayed by the instrument. In combination with your comments, we have supplemented this experiment in the experimental materials and methods section. The additional content is as follows :

SRB: The SRB we used was the mixed SRB cultured in laboratory. Mixed SRB was enriched from the wet soil in Haizhou open-pit coal gangue mountain in Fuxin City, Liaoning Province (121°41′E, 41°59′N) [24]. Wet soil was inoculated into sterile Starkey medium for anaerobic culture, which components were 0.5 g K₂HPO₄, 1.0 g NH₄Cl, 2.0 g MgSO₄·7H₂O, 0.5 g Na₂SO₄, 0.1 g CaCl₂·H₂O, 1.0 g yeast extract, 4 mL sodium lactate, 0.5 g (NH₄)₂Fe(SO₄)₂·6H₂O, 0.1 g ascorbic acid, 1 L distilled water, sterilizing for 30 min at pH=7, 121℃. Among them, (NH₄)₂Fe(SO₄)₂ and ascorbic acid can"t be sterilized at high temperature, so we used 0.22 μm filter membrane to filtrate and sterilize. When producing H₂S and emerging a large amount of black precipitates in the medium, it indicated that the mixed SRB has been cultured [24]. SRB in logarithmic growth phase was used to remove heavy metal ions by SRB. There were three replicates for each test setting. SRB was preserved in the method of glycerol tube cryopreservation, which was in sterile operating conditions, adding 500 μL bacterial solution and 500 μL 50 % glycerol solution into the sterilized 2 mL cryopreservation tube, mixing the solution lightly and preserving the mixed solution at -80°C.

Synthetic AMD: Taking the AMD from the mining area in Shanxi province to make on-site-survey. After measurement, we found that the pH of the AMD was 5±0.1, and there were 2.0±0.05 g/L SO₄^2-, 0.6±0.05 g/L Na⁺, 0.04±0.01 g/L Ca²⁺, 0.20±0.05 g/L Mg²⁺, 0.15±0.02 g/L K⁺，0.28±0.02 g/L Fe²⁺, 0.018±0.02 g/L Mn²⁺, 0.013±0.02 g/L Cu²⁺, 0.05±0.01 g/L Cr⁶⁺ in AMD. Combining with the measured AMD data and the components of the Starkey medium, we made artificial simulated AMD in laboratory. The pH value was adjusted by 1 mol/L HNO₃ and 1 mol/L NaOH to make the artificial simulated AMD pH=5. At the same time the artificial simulated AMD we made containing 2.0 g/L SO₄^2-, 0.58 g/L Na⁺, 0.04 g/L Ca²⁺, 0.20 g/L Mg²⁺, 0.17 g/L K⁺, and a small amount of Cl^-, HPO₄^2-, sodium lactate. To explore the removal efficiency of different heavy metals of Fe, Mn, Cu, Cr, we added different concentrations of Fe²⁺ into artificial simulated AMD to form AMD solution containing Fe²⁺ of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L. Adding different concentrations of Mn²⁺ into synthetic AMD to form 0 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Mn²⁺. Adding different concentrations of Cu²⁺ into synthetic AMD to form 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L AMD solution containing Cu²⁺. Adding different concentrations of Cr⁶⁺ into synthetic AMD to form 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L AMD solution containing Cr⁶⁺. FeSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O and K₂CrO₄ produced by Tianjin Zhiyuan Chemical Reagent Co., Ltd. were used to prepare the solutions containing Fe²⁺, Mn²⁺, Cu²⁺ and Cr⁶⁺ above. The above drugs are Analytical Reagent. The above solution was prepared with deionized water, and the ion contents in the above solution were measured by the instrument to avoid the error caused by the calculation.

Batch experiments were carried out to remove total Fe, total Mn, total Cu and total Cr by SRB. Among them, the test method of Fe removal by SRB: SRB at logarithmic growth stage was inoculated at 5% inoculum into AMD solutions containing Fe ion concentrations of 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L and 500 mg/L. Then 10 mL of sterilized liquid paraffin was added to the above mixture and the conical flask was sealed with a rubber stopper. The conical flask was placed in a constant temperature oscillation incubator (Changzhou Kaihang Instrument Co., Ltd., Changzhou, China, type HZ-9811K) at 35°C and 150 r/min for oscillation culture. There were three replicates for each test setting. Samples were taken at 1 d intervals. Using sterilised AMD without SRB inoculation as a blank group.The pH value, ORP value, OD₆₀₀ value and Ec value were measured by PHS-3C pH meter (Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China), CT-8022 ORP meter (Shanghai Hechen Energy Technology Co., Ltd. , Shanghai, China) and V-1600PC visible spectrophotometer (Shanghai Yuanwang Liquid Level Meter Co., Ltd., Shanghai, China). The samples to be tested were filtered with 0.22 μm filter membrane. The concentrations of SO₄^2- and total Fe in the filtered samples were measured by V-1600 PC visible spectrophotometer and Z-2000 flame atomic spectrophotometer (Hitachi society in Japan, Japan), and the removal percentage of SO₄^2- and total Fe were calculated. The average value and error of the three groups of repeated tests were used to draw the image. The experimental method of removing total Mn, Cu, Cr by SRB was consistent with that of removing total Fe by SRB.

The pH value was determined by electrode method (HJ 1147-2020). SO₄^2- was determined by barium chromate spectrophotometry (HJ/T342-2007). Total Fe and total Mn were determined by flame atomic absorption spectrophotometry (GB11911-89). Total Cu was determined by atomic absorption spectrophotometry (GB7475-87). Total Cr was determined by flame atomic absorption spectrophotometry (HJ757-2015).

The precipitation of SRB removing Fe²⁺ was collected and centrifuged at 4000 r/min for 10 min. The precipitation after centrifugation was dried at 90°C in a vacuum drying oven (DZF-6090AB). Then the dried precipitation was tested by SEM-EDS, XRD and XPS. The SEM appearance of the sample was detected by the German Zeiss sigma 500 scanning electron microscope, and the chemical substances on the surface of the precipitate were tested by EDS. The phase analysis was carried out by Bruker D8 Advance XRD, and the scanning step of XRD was 5°-90°. The XPS of precipitation was determined by Kratos AXIS SUPRA XPS.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The paper is now acceptable for publication.

Reviewer 2 Report

The revisions were generally acceptable but the English needs to be much improved. It's recommended to be editted by a commercial English editing company.

Reviewer 3 Report

The authors improved the manuscript after the first revision. Overall, the work is quite comprehensive, and quite competent, and technically looks ok. Even if I am not a native English speaker, I feel that there are still too many incorrect language details in the your manuscript, and further improvement are necessary to correct English to obtain high quality standards for this paper.

E.g. the first sentence in the abstract "Aiming at the problem of environmental pollution caused by heavy metals such as Fe, 10 Mn, Cu and Cr." has no predicate. 

Please avoid using very short sentences: "And the pH value was 4-6.4 [4]." - page 2, line 39, or "And the pH value was 3.5 [5]", page 2 , lines 41-42.

Reviewer 4 Report

If possible, add further clarification of the statistical analyses.