Biodegradation of Cyanide Using Soda Lake-Derived Alkaliphilic Microbial Consortia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript No. “IBB-D-24-00727”, submitted to “Water 

Title “Biodegradation of cyanide using soda lake-derived alkaliphilic microbial consortia”

 

Based on the comments below, this manuscript is accepted after minor revision.

Abstract:

1.     The abstract is lengthy and should be more concise, clearer and informative. The authors mentioned some information which should not be mentioned in the abstract “Residual cyanide was measured calorimetrically with a picric acid technique. Total ammonia, nitrite-nitrogen, and nitrate-nitrogen were measured with a digital photometer.” These two sentences are not critical to be mentioned in the abstract.

 

Keywords

2.     Make sure that the keywords used in the revised manuscript are not repeating the words used in the Title. In addition, keywords should be chosen better than they are.

 

Introduction

3.     Language of this paper needs improvement. Avoiding plagiarism should not affect the language of the manuscript.

4.     Uniform the terms in the manuscript.

5.     Authors should state the novelty of their work at the end of the introduction section, mentioning the need of the study.

6.     Authors should provide a comparison of current and past literature to highlight the significance of their work using recent citations.

7.     The introduction indicates an area of research but fails to specify a clear aim and objectives for the study. Please add these, correlate them to your methodology, and ensure that discussion and conclusions close the loop back to aim and objectives.

 

Materials and methods

 

8.     Is there any analysis or characteristics done for the inoculum sludge?

9.     Table 1: flow rate mentioned “0.83.33” this needs correction??

Conclusion

Author can add some significances of future work in conclusion section which make readers to get new direction for innovative ideas in the current field .

 

References

General comment: Out of 55 reference, no references were cited from the year 2024, only two from the year 2023, however, 75% of the references should be recently published articles not older than 5 years.

Comments on the Quality of English Language

 Language of this paper needs improvement. Avoiding plagiarism should not affect the language of the manuscript. Plagiarism percent of 39% is not accepted.

Author Response

Reviewer 1.

Based on the comments below, this manuscript is accepted after minor revision.

Authors response (AR): We thank the reviewer for the positive comment.

Abstract:

1. The abstract is lengthy and should be more concise, clearer and informative. The authors mentioned some information which should not be mentioned in the abstract “Residual cyanide was measured calorimetrically with a picric acid technique. Total ammonia, nitrite-nitrogen, and nitrate-nitrogen were measured with a digital photometer.” These two sentences are not critical to be mentioned in the abstract.

AR: Thank you for your valuable feedback. The authors appreciate the reviewer's suggestion and have made efforts to revise the abstract to be more concise and focused on the key aspects of the manuscript. The major changes are highlighted in the revised manuscript.

Keywords

2. Make sure that the keywords used in the revised manuscript are not repeating the words used in the Title. In addition, keywords should be chosen better than they are.

AR: In the revised manuscript, we have incorporated selected keywords that reflect the content and focus of the study by removing the previous keywords.

Introduction

3. Language of this paper needs improvement. Avoiding plagiarism should not affect the language of the manuscript.

AR: Thank you for this constructive feedback regarding the language of the paper. The authors have made significant improvements to enhance the clarity of the manuscript.

4. Uniform the terms in the manuscript.

AR: We accept the comment. The authors tried to use uniform terms.

5. Authors should state the novelty of their work at the end of the introduction section, mentioning the need of the study.

AR: In the last paragraph (Lines 67 to 69), we clearly outlined the gaps in previous studies and highlighted the novelty of our research, specifically presenting the integration of aerobic-anoxic reactors in series for this study and the use of alkaliphilic microbial consortia from a soda lake within an integrated treatment system

6. Authors should provide a comparison of current and past literature to highlight the significance of their work using recent citations.

AR: The authors include and compare the present study with previous studies and explain in the discussion part how much our study is effective relative to the previous studies.

7. The introduction indicates an area of research but fails to specify a clear aim and objectives for the study. Please add these, correlate them to your methodology, and ensure that discussion and conclusions close the loop back to aim and objectives.

AR:  Line (75-77): The authors appreciate the reviewer's suggestion to clarify the aims and objectives of the study. In the revised manuscript, we have explicitly outlined these elements and ensured they are well correlated with our methodology. Additionally, we have made the discussion, and conclusions effectively tie back to the stated aims and objectives, providing a cohesive narrative throughout the manuscript. The reviewer's comment has greatly enhanced the clarity and impact of our work.

Materials and methods

8. Is there any analysis or characteristics done for the inoculum sludge?

AR: The very important point for this study was cyanide and the pH. We measured the pH and salinity of the inoculum as indicated in the method part. The pH was 10.7 and salinity was 7.5 %.

8. Table 1: flow rate mentioned “0.83.33” this needs correction??

AR: This number does not appear in the Table. In the revised manuscript, we have addressed the typographical errors and made every effort to minimize any mistakes.

                                                Conclusion

9. Author can add some significances of future work in conclusion section which make readers to get new direction for innovative ideas in the current field .

AR: Line 387 to 389: The authors appreciate the suggestion, and the authors have added more significance of this study for future work to provide readers with new directions in the current field for the revised manuscripts. 

References

General comment: Out of 55 references, no references were cited from the year 2024, only two from the year 2023, however, 75% of the references should be recently published articles not older than 5 years.

AR: Thank you for your valuable feedback. We acknowledge the importance of including more recent references and have updated the manuscript accordingly. We have now incorporated additional references from the past five years, ensuring that most of the citations are from recent publications to reflect the current advancements in the field. We included 3 more new references that were published in the current year related to our study.

Comments on the Quality of English Language

 Language of this paper needs improvement. Avoiding plagiarism should not affect the language of the manuscript. Plagiarism percent of 39% is not accepted.

AR: We accept the comment We have carefully revised the manuscript to improve the language and ensure clarity, precision, and readability throughout. We appreciate your suggestion and believe the changes have strengthened the overall quality of the paper.

AR: We would like to clarify that the reported plagiarism percentage of 39% is incorrect. We have thoroughly checked the manuscript using reliable plagiarism detection software of our university, and the result showed above 94% originality. The authors have also made additional efforts to further minimize any potential similarities in the revised manuscripts.

 

Reviewer 2 Report

Comments and Suggestions for Authors

“ Biodegradação de cianeto usando consórcios microbianos alcalifílicos derivados de laca de soda ”

 

Comentários gerais

Este artigo aborda uma questão importante sobre a  remoção de cianeto de  águas residuais de minas de ouro usando microrganismos alcalifílicos com reatores aeróbicos e anóxicos conectados em série.  

 

Em geral, a estrutura do manuscrito segue as instruções dadas aos autores.

 

Revisão detalhada

-Algumas partes do texto precisam ser corrigidas ou melhor explicadas. Todos os detalhes da revisão estão no documento em anexo.

 

Avaliação final

 

Após a introdução das alterações sugeridas, acreditamos que o artigo pode ser publicado no Water  Journal.

 

Comments for author File: Comments.pdf

Author Response

Review 2.

This article addresses an important issue regarding the removal of cyanide from gold mine wastewater using alkaliphilic microorganisms with aerobic and anoxic reactors connected in series.

In general, the structure of the manuscript follows the guidelines provided to the authors.

Authors' response (AR): The authors acknowledge the reviewer's effort to give such detailed and constrictive comments, which helped us to improve the manuscript.

Detailed review

• Some parts of the text need to be corrected or better explained. All the review details are in the attached document.

AR: To facilitate our response, we have addressed the comments provided on the manuscript in the following document.

Final evaluation

After the suggested changes are implemented, we believe the article can be published in the Water Journal.

AR: The authors acknowledge the positive suggestion.

1. The meaning is not very clear. Please rewrite it.

AR: The sentence in the manuscript in the abstract section is rewritten based on the comment.

2. The reason why this type of reactor, aerobic followed by anoxic, was chosen must be indicated. The processes that can remove cyanide have not yet been mentioned.

AR: The authors indicated the importance of the integration of aerobic and anoxic reactors in the introduction. Cyanide can be biodegraded in a single reactor under specific culture conditions. However, high cyanide concentrations can inhibit denitrifiers, important for the treatment of reactive nitrogen molecules.

While previous studies focused on aerobic microbes, cyanide degradation can also occur in anoxic or microaerophilic environments. By integrating an anoxic reactor, the process becomes more efficient: the aerobic phase initiates cyanide degradation, while the anoxic phase handles reactive nitrogen species, enabling complete denitrification. This combination enhances microbial diversity and improves the overall treatment of various cyanide species, resulting in more effective removal and reduced toxic by-products

3. Please indicate the reasons why these cyanide concentrations were chosen

AR: The author acknowledges the comment, and in the revised manuscript, we have clarified and highlighted the amount of cyanide used for the initial treatment. It is noted that 200 mg/L is the threshold concentration of cyanide for common cyanide-degrading microorganisms.

4. 4. Please indicate where these pH values were measured

AR: It is indicated the pH was measured before the analysis of cyanide removal and it is the mean pH value collected from the aerobic and anoxic reactors from the sampling port of the reactors.

5. Please ensure that the chemical formulas of the compounds are written consistently. Review this

AR: The chemical formula we used in Table 2 is correct. In the optimization study, we switched the carbon source from sodium acetate to glycerol to determine if this change would impact the removal efficiency. This is the reason for the modification.

5. The equation presented depends on the equipment used and the standard concentrations used for the determination. I believe it can be removed.

AR: The authors acknowledge and accept the comment, and we removed the formula.

6. Please note that the origin of the various forms of nitrogen and how they are formed during the treatment process has not yet been mentioned in the text. This makes it difficult to understand the need to determine all these nitrogen compounds. The introduction should explain how cyanide removal occurs, along with the chemical reactions and biological processes involved. Since both aerobic and anoxic reactors are used, dissolved oxygen should also be measured.

AR: In the revised manuscript, the authors include the origin of nitrogen compounds from cyanide biodegradation, and it is highlighted in lines 52 to 55

7. Please specify at which stage of the treatment process it was added

AR: In the revised manuscript, the authors indicated sodium carbonate was added on days 7, 10, and 14. line numbers 247 to 248.

8. Please, the line type of the figure should be changed to make it easier to read in black and white

AR: The authors represented two parameters in the same figure, using black and white for better resolution of one parameter, and red to provide greater clarity for the other in comparison to black.

Please indicate at which point in the treatment system the sodium carbonate is added

AR: Sodium carbonate was added in both the aerobic and anoxic reactors and in the revised manuscript it is explained and highlighted in line 246

1. Please refer to the reactions that occur, the products that are formed, and the oxygen conditions to understand the discussion. Until now, the presence of enzymes has not been mentioned

AR: The authors accept the comment and modify it in the revised manuscript.

2. This point should be discussed considering information about the types of reactions and how cyanide degradation occurs. Please review this throughout the entire document

AR: The authors acknowledge the comment. However, since we used microbial consortia, we do not have a precise understanding of the reaction mechanisms involved. While we measured the compounds and confirmed their formation, the involvement of various organisms makes it challenging to indicate the specific mechanisms. If we had utilized a single microbial isolate, we would have been able to identify the mechanisms and the types of reactions occurring more definitively.

3. Please explain this better, mentioning how the cyanide removal reactions can affect the pH of the reactor.
4. Thank you for your comment. We have included a discussion of the factors that affect the pH of the reactor, such as ammonia production and acid production. The revised manuscript has been modified to reflect these changes for greater clarity in lines 367 and 368.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript provides a discussion on using biological treatment processes with source derived from Lake Chitu would be capable of treating cyanide-rich goldmine wastewater. The topic would be relevant in the fields of water, environment and sustainability. It is also commendable that the manuscript is generally well written.

However, there are a few points to address and that require revision to strengthen the manuscript.

 

1)

General comment: Using biological process to treat cyanide-rich wastewaters is not new and such treatment system has been implemented in practice. This study, however, could contribute by researching and documenting the process of acclimatising microorganisms and building up an effective microbial consortium for the treatment purpose. If this is indeed the objective, the authors may wish to revise the manuscript and clarify in the text accordingly.

Also, the manuscript should provide line numbers for ease of reference and to facilitate review.

 

2)

Page 3. Figure 1 and Table 1.

Please indicate in the schematic diagram how is sludge wasting carried out and whether carbon source is dosed in the anoxic reactor, etc.

More information should also be provided to characterise the wastewater – e.g. COD, TSS, conductivity, N, P, TDS, volume of air supply, DO level in the reactors, etc, and to confirm whether the reactor system is continuous rather than in batch.

Under this section, please also provide a table to summarise the parameters to achieve treatment goals – e.g. what exactly are the guidelines by WHO and ICMI in numbers.?

 

3)

Page 5. Section 3.1 with reference to alkaliphilic microbial consortia.

The photos provided may be inadequate. Please provide more information on the established microbial consortia – e.g. what were the common species found.?

 

4)

Page 5. Section 3.2 with reference to Cyanide degradation performance.

This section needs clarification. Figure 1 shows a continuous reactor system with a total HRT of 62.51 h – this seems to contradict what is written here – if the process is in batch or continuous.?

Please note that cyanide removed from the liquid stream does not necessarily mean that the cyanide is biodegraded, as some could be biologically adsorbed onto the sludge flocs.

The study should address and clarify to what proportion is cyanide biodegraded (converted to CO2) or bio-adsorbed on to the sludge flocs.

 

5)

Page 10. Section 4. Discussion. 1st Paragraph.

The text should justify (and avoid claiming) why this is a ‘novel’ treatment setup, as it is known that biological processes have been used for such treatment purpose – please also include a literature review to discuss on the different options to treat cyanide-containing wastewaters.

 

7)

Page 10. Section 4. Discussion. 2^nd Paragraph.

The text mentioned cyanide-degrading enzymes – what exactly are such enzymes, and may there be additional studies and information to quantify such enzymes.?

 

8)

Page 12. Section 5. Conclusion.  

After lab studies, what is the next step of development? – what is the roadmap to develop the technology on a larger scale and using real cyanide-containing wastewaters from actual goldmines?

 

 

Author Response

Reviewer 3.

Comments and Suggestions for Authors

The manuscript provides a discussion on using biological treatment processes with source derived from Lake Chitu would be capable of treating cyanide-rich goldmine wastewater. The topic would be relevant in the fields of water, environment and sustainability. It is also commendable that the manuscript is generally well written.

However, there are a few points to address and that require revision to strengthen the manuscript.

1. AR. Thank you for your positive and constructive feedback. We appreciate your acknowledgment of the relevance and quality of our manuscript. We also value your suggestions for improvement and we tried to address the points raised to strengthen the study further and greatly enhance the overall quality of the manuscript.

 

1)General comment: Using biological process to treat cyanide-rich wastewaters is not new and such treatment system has been implemented in practice. This study, however, could contribute by researching and documenting the process of acclimatising microorganisms and building up an effective microbial consortium for the treatment purpose. If this is indeed the objective, the authors may wish to revise the manuscript and clarify in the text accordingly.

Also, the manuscript should provide line numbers for ease of reference and to facilitate review.

AR: The authors agree with your observation that biological processes for treating cyanide-rich wastewater are not new. However, this study addresses three key aspects that were not the focus of previous research. First, it introduces a new treatment setup, consisting of aerobic-anoxic reactors connected in series and operated continuously, offering a more efficient approach to cyanide and cyanide intermediates treatment. Second, it employs highly alkaliphilic bacteria capable of surviving in extreme pH conditions (above pH 10) and effectively degrading cyanide. Third, the system is designed to treat cyanide and address cyanide derivatives, such as reactive nitrogen compounds. In this study, we successfully established and optimized the treatment system to enhance its overall effectiveness. 

 

Regarding the line numbers, we had initially included them for ease of reference, but unfortunately, the line disappeared maybe during the reviewer submission process, We apologize and thank you for your effort to address the point mentioned by paragraph number, table number, and figure numbers. 

2) Page 3. Figure 1 and Table 1.

Please indicate in the schematic diagram how is sludge wasting carried out and whether carbon source is dosed in the anoxic reactor, etc.

AR: In the revised manuscript, the authors have indicated the updated figure. However, the sludge was returned every 3 days by opening the end of the tube that connected to the clarifier. We indicated in the revised figure. where sludge was returned to the aerobic reactor. Regarding the carbon, during the simulated waste preparation, it was autoclaved separately but later mixed with other components. The waste was continuously fed into the aerobic reactor, with partially treated waste flowing continuously to the anoxic reactor. The waste in the anoxic reactor also contained carbon.

 

 

More information should also be provided to characterise the wastewater – e.g. COD, TSS, conductivity, N, P, TDS, volume of air supply, DO level in the reactors, etc, and to confirm whether the reactor system is continuous rather than in batch.

 

Under this section, please also provide a table to summarise the parameters to achieve treatment goals – e.g. what exactly are the guidelines by WHO and ICMI in numbers.?

 AR: The authors have measured mainly TAN, TIN, Nitrite nitrogen, nitrate nitrogen, and pH. In our next pilot-scale study, this is a very important input, and we acknowledge this.

 

3) Page 5. Section 3.1 with reference to alkaliphilic microbial consortia.

The photos provided may be inadequate. Please provide more information on the established microbial consortia – e.g. what were the common species found.?

 AR: We used microbial consortia for the acclimatization process, without any isolation of individual strains. The goal was to establish a cyanide-degrading consortium and develop the treatment system. Currently, with the optimized treatment system, we are conducting a pilot-scale study, which includes identifying the key microbial components involved in the treatment process.

4)Page 5. Section 3.2 with reference to Cyanide degradation performance.

This section needs clarification. Figure 1 shows a continuous reactor system with a total HRT of 62.51 h – this seems to contradict what is written here – if the process is in batch or continuous.?

AR: The treatment system operated continuously (the setup was continuous, connected in series), and we calculated the average residence time of the waste in the reactor before the partially treated wastewater exited the clarifier. The hydraulic residence time in this case refers to the amount of time the waste remains in the aerobic and anoxic reactors before leaving the reactors. In a batch treatment system, treatment duration is often referred to as "treatment days," or simply the time required for the process to complete. However, in a continuous flow system, hydraulic retention time (HRT) is the term used to describe the average time the liquid waste stays in the reactor. Treatment days in a batch system refer to the total time for a complete treatment cycle, while HRT in a continuous system measures the average time the waste is spent in the reactor.

Please note that the cyanide removed from the liquid stream does not necessarily mean that the cyanide is biodegraded, as some could be biologically adsorbed onto the sludge flocs.

AR: Thank you for the valuable input and we considered this as part of our discussion.

The study should address and clarify to what proportion is cyanide biodegraded (converted to CO2) or bio-adsorbed on to the sludge flocs.

 AR: Cyanide is broken down into less harmful compounds such as ammonia, formate, or even CO2. However, the complete conversion of cyanide to CO₂ is not always guaranteed or the dominant pathway, as this depends on the specific microbial pathways involved. As indicated in the methods, our inoculum is a microbial consortium, consisting of various microorganisms that can convert cyanide into different intermediates. If we had used a single microbial isolate, it would have been possible to demonstrate the degradation mechanism and predict the final products. The authors acknowledge the comment and consider it as input for the later study.

 

5)Page 10. Section 4. Discussion. 1st Paragraph.

The text should justify (and avoid claiming) why this is a ‘novel’ treatment setup, as it is known that biological processes have been used for such treatment purpose – please also include a literature review to discuss on the different options to treat cyanide-containing wastewater.

AR: We acknowledge the comment and have revised the paragraph accordingly. In the updated manuscript, the term "novel" has been changed to "new." Various treatment approaches for cyanide-contaminated wastewater have been explored in the past. In our study, we propose a new treatment setup that integrates both aerobic and anoxic reactors in series. Previous studies have predominantly focused on aerobic treatment of cyanide waste; however, for complete cyanide removal, it is essential to incorporate an anoxic reactor. This creates the necessary conditions for the denitrification process, which aids in treating reactive nitrogen compounds produced during cyanide degradation. 

7)Page 10. Section 4. Discussion. 2^nd Paragraph.

The text mentioned cyanide-degrading enzymes – what exactly are such enzymes, and may there be additional studies and information to quantify such enzymes.?

1. AR. We accept the comment. In the revised manuscript, we improved the sentence.

8)Page 12. Section 5. Conclusion.  

After lab studies, what is the next step of development? – what is the roadmap to develop the technology on a larger scale and using real cyanide-containing wastewaters from actual goldmines?

AR: Thank you for your feedback. This project focuses on treating wastewater from gold mining. The current phase is the first step, aimed at testing whether the treatment setup is effective and optimizing the system. The next step will involve scaling up the treatment system and repeating the process using actual wastewater, following the same treatment setup. This will be done before proceeding to a large-scale study.