An In Vitro Study of the Effects of Temperature and pH on Lead Bioremoval Using Serratia marcescens

Round 1

Reviewer 1 Report

Abstract:

1         “A design was adopted using pH (3.5 and 7), temperature (25, 30, and 35 °C), and 24 hours of incubation as independent variables. The inoculum of 108 cfu/ml of Serratia marcescens was constant in all treatments. Different treatments were evaluated after 24 hours of incubation”

1.1   pH (3, 5 and 7)

1.2   Please remove “24 hours of incubation” from the list of independent variables.

1.3   Is it 108 cfu/ml or 10^8 cfu/ml?

 

2         “The results show that treatment 6 (temperature 35°C, 5 × 108 CFU/mL, and pH 5) showed more excellent lead bio removal after 24 hours of incubation, giving a removal percentage of 63.9%.”

2.1   Again is it 10^8 or 108?

2.2   Replace “more excellent” with “excelent”

2.3   Why authors think 63.9% removal is excellent removal? How does it compare with other reports?

2.4   What was the CFU count after 24?

 

Introduction

3         “the Yellow River Basin has the high- 49 est levels of lead (Pb) than other basins, which is found”

3.1   Remove “which is found” and replace “,” with “.” after the waord basins.

4         Please write the microorganism names in italics wherever applicable

5         Line no 121: “First, I observed” – correct this

6         Line 131: “a potential cathodic current of 900 Mv,” please cross check and correct this. should be “a cathodic potential of 900 mV”

7         Line 134: “I developed the research by applying.. “ correct this

Materials and methods:

8         Line 150: “design”

9         Line 186: 10 ppm

10     Line 192: pH 3.5 à pH 3, 5

11     Line 194: Why CFU 108/mL is chosen?

12     Table 1 & 2 do not match

13     Table 2. What is Effluent 20%?

13.1           Place the footnote below the table

14      Line 217: better to use equation editor

15     Line 220: add units for Cf also

16     Results for pH 3 is not shown in the manuscript.

17     Figure 2 does not provide additional information. Can be removed

18     Figure 3. Triplicates average with std dev or error bar should be plotted rather than individual plots like this

please refer above

Author Response

Dear colleague, I hope you are in good health.
The authors appreciate the corrections and suggestions made; and house one have been answered:

1         “A design was adopted using pH (3.5 and 7), temperature (25, 30, and 35 °C), and 24 hours of incubation as independent variables. The inoculum of 108 cfu/ml of Serratia marcescens was constant in all treatments. Different treatments were evaluated after 24 hours of incubation”

1.1   pH (3, 5 and 7)

1.2   Please remove “24 hours of incubation” from the list of independent variables.

1.3   Is it 108 cfu/ml or 10^8 cfu/ml?

Ans. It was corrected. Only pH 5 and 7 were tested. It was mistaken in the writing, which was corrected.

2         “The results show that treatment 6 (temperature 35°C, 5 × 108 CFU/mL, and pH 5) showed more excellent lead bio removal after 24 hours of incubation, giving a removal percentage of 63.9%.”

2.1   Again is it 10^8 or 108?

2.2   Replace “more excellent” with “excelent”

2.3   Why authors think 63.9% removal is excellent removal? How does it compare with other reports?

2.4   What was the CFU count after 24?

 Ans. It was corrected.

Introduction

3         “the Yellow River Basin has the high- 49 est levels of lead (Pb) than other basins, which is found”

3.1   Remove “which is found” and replace “,” with “.” after the waord basins.

Ans. It was corrected. The sentence was rewritten: “A variety of factors have led to the contamination of other water bodies with heavy metals over the last decades; for instance, in China, the Yellow River Basin contains the highest levels of lead (Pb) compared to other basins, which are closely linked to the development of these regions' economies and industrial sectors“.

4         Please write the microorganism names in italics wherever aplicable

Ans. It was corrected in all documents.

5         Line no 121: “First, I observed” – correct this

Ans. It was corrected, it was a written mistake.

6         Line 131: “a potential cathodic current of 900 Mv,” please cross check and correct this. should be “a cathodic potential of 900 mV”

Ans. It was corrected: “The conditions evaluated in this investigation were a Cu (II) concentration of up to 80 mg/L, a potential cathodic of 900 mV, and a standard hydrogen electrode”

7         Line 134: “I developed the research by applying.. “ correct this

Ans. It was corrected. The sentence was modified.

Materials and methods:

8         Line 150: “design”

Ans: changed to "Experimental design"

9         Line 186: 10 ppm

Ans. It was corrected.

10     Line 192: pH 3.5 à pH 3, 5

Ans. The pH 3 was a mistake.

11     Line 194: Why CFU 108/mL is chosen?

12     Table 1 & 2 do not match

Ans. It was corrected. Table 2 was deleted.

13     Table 2. What is Effluent 20%?

13.1           Place the footnote below the table

Ans. Table 2 was deleted. The contents of the table were written.

14      Line 217: better to use equation editor

Ans. lead removal % = (C₀ – C_f / C₀) ? 100   In this way we place the equation as the magazine requires.

15     Line 220: add units for Cf also

Ans: the unit was placed, mol/L.

16     Results for pH 3 is not shown in the manuscript.

Ans. The pH 3 was deleted, it was a mistake of writing.

17     Figure 2 does not provide additional information. Can be removed

Ans. All figures were corrected and changed.

18     Figure 3. Triplicates average with std dev or error bar should be plotted rather than individual plots like this

Ans. It was added.

 

kind regards

Author Response File: Author Response.pdf

Reviewer 2 Report

Thank you very much for inviting me to review the manuscript " Influence of temperature and pH on lead bioremediation using 2 Serratia marcescens ”. This manuscript aimed to investigate the potential of Serratia marcescens bacterium in removing lead from contaminated water. The strengths of the research lie in its focus on bioremediation as an alternative method for lead removal, the introduction of a potential lead-removing bacterium, and the systematic exploration of different physicochemical parameters. The manuscript is interesting, but from my point of view, this requires some changes and explanations should be made previously that the manuscript will be published in Sustainability.

 

1. The present manuscript contains too much typo and grammatical mistakes Please read carefully and correct it. For example:

Lines 30, 146, 154, 234, 236, 287, 307, etc. extra space or extra space or hyphen omitted: bio removal.

Lines 25, 129, 146, 184, 187, etc. the scientific name of the bacterium is not in italics.

Line 183 Spanish word “de”.

Line 41, 50, 113, 130, 182, etc. inconsistent use of commas and other punctuation marks.

No chemical formula in the manuscript has subscripts.

Lines 255, 257: liter symbol written in lowercase.

Line 191 and 192: inconsistent sentence.

2. The references and bibliography do not conform to the journal's format, please correct them.

3. The abstract should be rewritten by summarizing the problem, the method, the results, and the conclusions.  

4. The authors should consider some representative word in the keywords. Some less prominent key words could be replaced by significant word.

5. The introduction should be rewritten due to the following reasons:

The introduction initially focuses on the presence of heavy metals in rivers exclusively in China. However, according to the “2.2. Sample collection” section, the samples were collected from a river in Peru.

Introduction should provide more context about the specific heavy metal contamination issues in Peru.

No other lead or heavy metal removal methods are mentioned in the introduction.

It is not mentioned why the bioremediation method is used and why the bacteria Serratia marcescens is used, what are its advantages and disadvantages?

6. Section "2. Materials and Methods" should describe the technical data and all research methods. Section 2 should describe how the pH was determined, if instrumentally, then indicate the instrument. The initial conditions of pH and temperature of the samples are not indicated, as well as the reagents used to modify the pH. Why are only 6 of the 9 treatments carried out in Table 3?

7. The research could benefit from a larger sample size to increase the statistical power and generalizability of the findings.

8. The methodological inaccuracies include the absence of specific details about the water sample preparation, bacterial culture conditions, and the precise method used to measure lead levels. These details are crucial for replicating the experiment and validating the results. Furthermore, the paper lacks proper controls, such as a negative control group without the Serratia marcescens bacterium, to ensure that observed lead removal is solely attributable to the bacterial activity.

9. The testability of the hypothesis could be improved by providing more comprehensive data, including replicates and error bars, to assess the robustness and reliability of the results.

10. The text could benefit from presenting the experimental results in a more organized and structured manner. Consider using tables, graphs, or figures to clearly illustrate the data and make it easier for readers to comprehend the findings at a glance.

11. You should provide more information on the statistical methods used to analyze the data and determine the significance of the results. Mention the specific tests or models employed and include relevant statistical parameters, such as p-values or confidence intervals, to support the conclusions drawn from the data.

 

Authors should check the grammatical and english errors. I suggest authors to proof-editing to the entire manuscript, it will significantly help to improve English language.

Author Response

Dear colleague, I hope you are very well.
The authors appreciate the comments made, the same ones that were corrected, in the following parts:

1. The present manuscript contains too much typo and grammatical mistakes Please read carefully and correct it. For example:

Lines 30, 146, 154, 234, 236, 287, 307, etc. extra space or extra space or hyphen omitted: bio removal.

Ans. It was corrected. Many parts of the manuscript were modified.

Lines 25, 129, 146, 184, 187, etc. the scientific name of the bacterium is not in italics.

Ans. It was corrected.

Line 183 Spanish word “de”.

Ans. It was corrected.

Line 41, 50, 113, 130, 182, etc. inconsistent use of commas and other punctuation marks.

Ans. It was corrected.

No chemical formula in the manuscript has subscripts.

Ans. It was corrected.

Lines 255, 257: liter symbol written in lowercase.

Ans. It was corrected.

Line 191 and 192: inconsistent sentence.

Ans. The sentences were modified.

2. The references and bibliography do not conform to the journal's format, please correct them.

Ans. It was corrected.

3. The abstract should be rewritten by summarizing the problem, the method, the results, and the conclusions.  

Ans. The abstract was rewritten.

4. The authors should consider some representative word in the keywords. Some less prominent key words could be replaced by significant word.

Ans. Some keywords were changed: Serratia marcescens, lead, bioremoval, heavy metals, bioremediation.

5. The introduction should be rewritten due to the following reasons:

The introduction initially focuses on the presence of heavy metals in rivers exclusively in China. However, according to the “2.2. Sample collection” section, the samples were collected from a river in Peru.

Introduction should provide more context about the specific heavy metal contamination issues in Peru.

No other lead or heavy metal removal methods are mentioned in the introduction.

It is not mentioned why the bioremediation method is used and why the bacteria Serratia marcescens is used, what are its advantages and disadvantages?

 

Ans. It was corrected

6. Section "2. Materials and Methods" should describe the technical data and all research methods. Section 2 should describe how the pH was determined, if instrumentally, then indicate the instrument. The initial conditions of pH and temperature of the samples are not indicated, as well as the reagents used to modify the pH. Why are only 6 of the 9 treatments carried out in Table 3?

 

Ans. It was corrected

7. The research could benefit from a larger sample size to increase the statistical power and generalizability of the findings.

Ans. It was corrected

8. The methodological inaccuracies include the absence of specific details about the water sample preparation, bacterial culture conditions, and the precise method used to measure lead levels. These details are crucial for replicating the experiment and validating the results. Furthermore, the paper lacks proper controls, such as a negative control group without the Serratia marcescensbacterium, to ensure that observed lead removal is solely attributable to the bacterial activity.

Ans. Added more details to the methodology.

9. The testability of the hypothesis could be improved by providing more comprehensive data, including replicates and error bars, to assess the robustness and reliability of the results.

Ans. It was add.

10. The text could benefit from presenting the experimental results in a more organized and structured manner. Consider using tables, graphs, or figures to clearly illustrate the data and make it easier for readers to comprehend the findings at a glance.

Ans. The result was reorganized. It included new figures.

11. You should provide more information on the statistical methods used to analyze the data and determine the significance of the results. Mention the specific tests or models employed and include relevant statistical parameters, such as p-values or confidence intervals, to support the conclusions drawn from the data.

Ans. It was corrected. It was used The Tukey test for the comparison of the Total Pb values.

kind regards

Author Response File: Author Response.pdf

Reviewer 3 Report

The reviewer's comments are attached in a separate Word document

Comments for author File: Comments.pdf

English must be carefully examined and polished once more.

Author Response

Dear colleague, I hope you are very well.
The authors appreciate the comments made, the same ones that were corrected, in the following parts:

Comments and suggestions for the author
The manuscript titled "Influence of temperature and pH on lead bioremediation" using Serratia marcescens" submitted by D. Lecca-Caballero et al. can be considered for publication in sustainability Journal, after a major review. The present work has its importance and applicability in the field of biological lead removal. The article is Interesting for scientists relevant to this area, but major modifications are needed before the manuscript can be accepted for publication in the Sustainability journal."
The following comments will help increase the quality of the manuscript before publication. Below are comments and suggestions that need to be addressed.
1. Authors are suggested to refine a new manuscript title. The title is not accurate.
Answer: Changed to An in vitro study of the effects of temperature and pH on lead removal using Serratia marcescens
2. The abstract should briefly state the purpose of the research, the main results, and important conclusions. Authors must rewrite the abstract for these standards.
Answer: It was improved based on your observations.
3. How is this work different from others and how new is this study? possess? The author has to work on this and show us why this work is useful for generalized applications.
Ans. It seeks to optimize the conditions in which our Serratia marcescens strain has the capacity to bioremediate water contaminated with Pb in natural sources of ecosystems in Peru. In addition, it will allow the creation of a pilot plant in the mining sector to bioremediate water with Pb that converge in this economic activity, in such a way, to contribute to reduce the impact of lead contamination and to reuse the water resource for other economic activities. .

4. The authors should indicate the ideal content of lead in the effluent (according to the standard value of the Pollution Control Board) and should provide information on removal efficiency.
Ans. The Council of Ministers through SUPREME DECREE No. 004-2017-MINAM establishes the annual value of minimum lead concentration in rivers of the Coast and Sierra of 0.05 mg/L. (mentioned in handwritten)
https://sinia.minam.gob.pe/normas/establecen-valor-anual-concentracion-plomo
5. The manuscript should emphasize the importance of this work and detail how Serratia marcescens can be used on a large scale in real time?
Ans. Bioremediation is an effective alternative to maintain sustainable environments; Likewise, it is important to use this process in the treatment of water contaminated with heavy metals to reduce the concentration of lead in particular to acceptable levels through the use of the S. marcescens bacterium, thus contributing to the decontamination of our ecosystems.

6. The justification why the heavy metal lead and Serratia marcescens were selected for this study. The authors should explain the importance of the heavy metal above. and microbes other than the reported materials.
Ans. Lead has been considered for decades one of the most important environmental contaminants. It is known as an environmental carcinogen, in addition, it is a toxin that is defined as cumulative, for which reason there is increasing evidence of its link in the pathogenesis of chronic diseases.
http://www.scielo.edu.uy/scielo.php?script=sci_arttext&pid=S1688-03902006000400005
For the use of a microorganism, certain characteristics are required that they must have: Resistance and adaptability. Therefore, the use of Serratia marcescens is due to the fact that this bacterium stands out as it is part of isolates of bacteria resistant to heavy metals. In addition, that allows easy adaptation to places with high concentrations of metals.

7. Authors should make the font italic in Serratia marcescens on line no 146.
Ans. It was corrected

8. Authors should add the detailed procedure for the isolation of Serratia
Marcescense culture with appropriate references. What pH is maintained for the preparation of the media?
Ans. The Serratia marcescens strain is part of the collection of the Research Institute of the Cesar Vallejo University, so it went through all the identification controls..

9. The author must incorporate the schematic representation for the preparation of the Serratia marcescens strain.
Ans. The Serratia marcescens strain was characterized by the Research Institute using the Vitek II microbiological identification system and in this study we limited ourselves to performing some biochemical tests to determine the activity and purity of the strain.

10. Authors should review the lead removal efficiency equation in line #217. That is not correct.
Ans. It was corrected

11. Authors should explain the effect of pH on lead removal. A detailed and
a proper explanation of the effect of pH is needed in section 3.2
Ans pH is directly related to the ability of hydrogen ions to compete with metal ions on the biosorbent surface, resulting in better removal rates.

12. Authors should study the effect of other parameters on heavy metal lead removal from the aqueous phase and incorporate RSM studies for the
Optimization of various process parameters to achieve removal efficiency.
Ans. It is a recommendation that will be taken into account in future research.

13. Authors must incorporate the equation of the regression model (response
variable) from statistical studies for lead removal.
Ans. It is a recommendation that will be taken into account in future research.

14. Optimization studies must be supported by the following references and
should add in the revised manuscript
https://doi:10.5004/DWT.2018.21799
https://doi.org/10.3390/chemengineering7020031
Ans. It is a recommendation that will be taken into account in future research.

15. Authors should compare the specific experimental data obtained with the
Results of the authors of other works on lead bioremediation
Ans. It was corrected

16. Authors should incorporate the detailed mechanism (schematic representation) for lead removal using Serratia marcescens
Ans. Stomara into account in future research.

17. Authors should verify lead removal from actual industry heavy metals
effluent for the viability of the Serratia marcescens strain. (Authors should verify
applications of the Serratia marcescens strain in real industrial effluents)

Ans. The sample used in the investigation was from an actual lead-contaminated effluent; product of mining activity. Likewise, this test was carried out under laboratory conditions taking into account parameters such as temperature, Ph, inoculum concentration so that bioremediation can be carried out and have its practical application on a large scale in the future.

18. English must be carefully examined and polished once more.
Ans. It was corrected

19. The authors will replace the obsolete references with the most recent ones.
(after 2018). in order to update research in this area.

Ans. It was corrected

Best regards

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

microbial species should be written in italics throughout

check the language improve the language

language can be improved

Author Response

Dear colleague, I hope you are well.
The authors have corrected the errors mentioned. I hope the manuscript is to your liking.
kind regards

Author Response File: Author Response.pdf

Reviewer 2 Report

the authors have made the changes suggested by the reviewer and the manuscript can now be accepted for publication.

Extensive editing of English language is required

Author Response

Dear colleague, I hope you are well.
The authors have corrected the errors in English. I hope the manuscript is to your liking.
kind regards

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors addressed all the comments appropriately. Now the manuscript can be accepted for publication in the Journal "Sustainability"

Author Response

Dear colleague, I hope you are well. The authors appreciate his comments. kind regards

Author Response File: Author Response.pdf