Efficacy of Enzymatically Induced Calcium Carbonate Precipitation in the Retention of Heavy Metal Ions

Round 1

Reviewer 1 Report

The article was prepared in accordance with the requirements of a scientific article published in Sustainability.

It presents an interesting results of the efficacy of Enzyme Induced Calcite Precipitation (EICP) in the context of the mobility of heavy metals in soils. 

Comments as below:

1. Introduction should be rewritten. It should contain the introduction to the topic of the paper (usually called background), research gap/ question and description of the authors answer to the research gap/ question. The literature review (here indicated as background) should be a separate section, or this should be removed from the title "Introduction & Background"
2. The tablets are not prepared in accordance with the requirements of the journal - please correct
3. There is lack of deep discussion of the results.
4. the strong connection with the sustainability should be considered and discussed here. How will this method improve the quality of the environment/ where it can be widely used, etc.

Author Response

The authors thank the reviewer(s) for their constructive comments. The authors have addressed all the comments and marked them in the revised manuscript using RED Color Font. Note: The entire manuscript has been proofread by an English native speaker and checked for consistency in grammar, punctuation and mistakes. The page numbers which have been referred by the reviewer correspond to first revision. In the revised version, due to addition of new sentences as well as comprehensive editing undertaken, the respective line numbers would have changed. The author(s) have responded to the queries in this response sheet relying on the same line numbers.  

1. Introduction should be rewritten. It should contain the introduction to the topic of the paper (usually called background), research gap/ question and description of the authors answer to the research gap/ question. The literature review (here indicated as background) should be a separate section, or this should be removed from the title "Introduction & Background".

Response: The introduction part has been rewritten. The research gap is addressed as “to explore a sustainable method of contaminant remediation and replace the conventional methods of soil stabilisation”. Literature referred here is separated in the introduction part. Background part is separated and provided as separate section.

2. The tables are not prepared in accordance with the requirements of the journal - please correct

Response: They have been now revised. The tables are prepared according to the requirements of the journal.

3. There is lack of deep discussion of the results.

Response: The results are organised according to the contaminant spiked in the soils and presented for better understanding. The author(s) believe that every article has its own way of taking the discussions pertaining to results obtained.

4. The strong connection with the sustainability should be considered and discussed here. How will this method improve the quality of the environment/ where it can be widely used, etc.

Response: The authors agree with the reviewer. Accordingly, the following sentences have been incorporated in the introduction part to address the issue pertaining to sustainability and relating the same to the present study.

Stabilization of soil by cement and lime have been vastly used [34–37], and contribute to emission of greenhouse gases leading to large scale global warming. Production of cement and lime result in about 800-900 kg and 600-700 kg CO₂ respectively per ton [38]. Cement industry contributes about 5% of CO₂ emissions globally [39,40]. With such an alarming situation, researchers have examined for sustainable stabilization methods for soil wherein partial replacement or total replacement of the cement or lime binders are tested for their reliability. These stabilizers include palm oil fuel ash [41], flyash [42], rice husk ash [43,44], residue of calcium carbide [45], alkali-activated agro-waste [46] etc. As an attempt to contribute to the field of soil stabilization bio cementation by CaCO₃ is also employed as a sustainable method of soil stabilization [47–51] which includes improvement of geotechnical properties of soil as well as contaminant remediation.

Author Response File: Author Response.docx

Reviewer 2 Report

The paper’s title match its content. The Introduction and Background section is sufficient. The research topic presented clearly, aim of article clearly specified and realized. The article have a logical layout. The language of article correct. The paper’s conclusions follow logically from the development of the argument. The text adequately illustrated. 

A few comments for the Authors:

1. Line 142-145: For "K" and "M" soils (e.g. in brackets) please refer to the soil types according to WRB 2015.
2. Line 155: What does AR mean? Please explain the abbreviation.
3. Line 170: What does ESs mean? Please explain the abbreviation.
4. Tables 3 and 2 are in reverse order.
5. In Table 2: What does unit "M" mean? 
6. Line 231: What does AAS mean? Please explain the abbreviation.

Author Response

The authors thank the reviewer(s) for their constructive comments. The authors have addressed all the comments and marked them in the revised manuscript using RED Color Font. Note: The entire manuscript has been proofread by an English native speaker and checked for consistency in grammar, punctuation and mistakes. The page numbers which have been referred by the reviewer correspond to first revision. In the revised version, due to addition of new sentences as well as comprehensive editing undertaken, the respective line numbers would have changed. The author(s) have responded to the queries in this response sheet relying on the same line numbers.  

• Line 142-145: For "K" and "M" soils (e.g. in brackets) please refer to the soil types according to WRB 2015.
• Response: Agreed. The types mentioned in WRB were referred. K indicates Kaolinitic (Red Soil) type and M refers to Montmorillonite type soils.
• Line 155: What does AR mean? Please explain the abbreviation.
• Response: Agreed. AR means “Analytical reagent”, it has been incorporated in the manuscript as “Analytical reagent grade nitrates of nickel (Ni(NO₃)₂), cadmium (Cd(NO₃)₂), and lead (Pb(NO₃)₂) were used to prepare the stock solutions.”
• Line 170: What does ESs mean? Please explain the abbreviation.
• Response: ESs is abbreviated in Line 29 that is in Abstract as Enzyme solutions
• Tables 3 and 2 are in reverse order.
• Response: Agreed. Thanks for pointing this out. These tables are named properly now.
• In Table 2: What does unit "M" mean?
• Response: M stands for Molarity.
• Line 231: What does AAS mean? Please explain the abbreviation.
• Response: AAS stands for Atomic absorption spectrophotometer. It has been mentioned in section 2.2 as “Atomic absorption spectrophotometer (AAS)”.

Author Response File: Author Response.docx

Reviewer 3 Report

This is an interesting paper discussing the “Efficacy of Enzymatically Induced Calcium Carbonate Precipitation in the Retention of Heavy Metal Ions”.

First of all, I suggest to revise and check English language all through the text (English native speaker is recommended), especially vocabulary and structure of sentences because there are many linguistic inaccuracies (mistakes) that sometimes make difficult the understanding of the research.

From a general point of view, I think that the paper has two great weak points:

• the authors have reported experimental results in tables and figures without standard deviations. It is not possible to present data with statistical significance without this information. It is, hence, mandatory to add this information both in the tables and in the figures. Moreover, even if I think that the figures are more understandable and interesting than the tables, I suggest to show the data also in tables reported in Supplementary Material to evidence better the observed trends. However, it is always necessary to report in tables not only average values but also the respective standard deviations. Moreover, the authors should also report pH values found in several experiments as they themselves repeatedly declare the importance of this parameter on the mobility of metals in a soil.
• thе manuscript contains a large set of measurements; however, the experimental data have not been statistically processed to evaluate the statistically significant differences between the compared values. I certainly believe that a statistical approach (for example the ANOVA treatment) would substantially improve this work, and therefore I encourage the authors to conduct it.

The other criticisms of the work are:

Abstract: It is necessary to explain already in this paragraph how enzyme solutions (1, 2 and 3) differ. This information is fundamental to understand the choice to use more enzyme solutions.

Lines 41-45: The meaning of these two sentences is the same. I suggest to the authors to combine them so as not to repeat the same concept twice.

Line 45: I propose to change “seizing” with “decreasing”.

Lines 45-46: In this sentence there is not a verb. It is necessary to add it.

Introduction & background: The introduction is poorly structured and often strenuous to read due to the excessive number of short paragraphs. I suggest to the authors to review carefully the text with the aim to provide greater linearity and organicity to the presented studies.

Lines 64-65: The high level of toxicity of leachates is due to the presence of heavy metals, etc. In my opinion it is necessary to clarify this concept by changing this sentence.

Lines 68-71: This sentence is not clear. The presence of less than 1000 mg/kg of heavy metals in soil is less problematic than what? In this sentence “heavy metals” are repeated three times and this makes the sentence excessively long and redundant. It is possible to change this sentence in order to make it more fluent and understandable.

Lines 73-75: The industrial activities can increase the heavy metals concentrations in soils, but the reason of their contribution is mainly due to their industrial effluents with respect to their atmospheric emissions. The authors should report both these sources.

Lines 79-80: It amazes me that there is only one reference reported for this statement. In my opinion the authors should add more references.

Lines 86-87: This sentence is not clear. The authors should rewrite it paying more attention to the concept they want to expose. In particular, the heavy metals assimilation in plants is a problem because this determines that the pollutants can be introduced in the food chain. This concept must be clearly stated.

Lines 88-89: It is not possible to start a sentence with “And”. Moreover, what is meant by “chemical base”? The authors should explain it. Finally, in this sentence there is not the point.

Lines 89-90: I suggest to add “for example” in this sentence because the authors do not report all damages for human health due to the high concentrations of Hg, Pb and Cu. Moreover, I think there is a mistake because the potentially toxic element which causes bones damage is Cd and not Cu, as reported by the authors.

Materials and methods: In this paragraph the chemical-physical parameters of the two soils are completely missing. It is absolutely necessary to report at least soil pH, organic carbon content, Ca, Cd, Ni and Pb concentrations.

Line 169: The table reporting the Cd, Ni and Pb concentrations determined in treated and untreated soils is Table 2 and not Table 3. Please, correct this mistake.

Line 171: The contaminants are three. Please, write “contaminants” in place of “contaminant”.

Line 182: It is necessary to change “Table 3” with “Table 2”.

Line 184 and line 189: Please, change “table 2” with “table 3.

Line 205: I suggest to specify that the amount of urease enzyme in ES3 is lower than in ES2 because now this concept is not clearly reported in this sentence.

Line 212: The authors must add the information regarding soaking time with 1 M HCl and drying temperature.

Lines 219-221: I do not understand because the authors write that the extractants remove heavy metals “retained in the soil by acid digestion”. The acid digestion permits to extract quantitively heavy metals by solid samples, not to retain them.

Lines 219-221: For a correct evaluation of extractability of heavy metals from the soil, the authors should also have used DTPA-TEA extraction solution as extractant because it is one of the main extractants used for this purpose.

Lines 243-244: In my opinion it is necessary to explain better the effect of urease enzyme on CaCO₃ precipitation mechanism since all the work done is based on the efficiency of this mechanism.

Lines 266-267: In this sentence there is not a verb. Please, add it.

Figure 2, 3 and 4: Table and figure captions must be self-explanatory. Thus, the caption of figures should indicate at least what was reported in each box (a, b, c and d).

Lines 269-274: I do not understand the connection among pH, extractant concentration and type of extractant on Cd retention behavior in the soil. I think that the presence of citric acid decreases pH in the soil and this causes a lower Cd retention with increasing of citric acid concentration but this must be demonstrated by pH values which are now not reported in the manuscript.

Lines 279-280: How the authors can affirm that the difference “in removal efficiency was not significant between the two load ratios” if they have not statistically processed the data obtained and standard deviations are not known?

Line 282: It is necessary to add “for M and K soil respectively” after “72.36–57.2%”.

Line 311: The information reported in this sentence is not correct because it is visible from figure 4 that the removal efficiencies were 26.26 % and 11.2 % for the K soil with 50 mg/kg and 100 mg/kg load ratios, respectively. Hence a higher load ratio determines a greater effectiveness of the EICP treatment.

Lines 314-315: This sentence is not clear. I think that Pb and Cd can be adsorbed on CaCO₃ and this determines their greater retention in the soil.

Lines 319-320: Does lead precipitate as hydroxide or as sulfate? Please explain it because this is not clear in the sentence.

Lines 327-333: It is absurd that the authors discuss the effect of pH on heavy metals retention mechanisms in the soil but do not report these data in the manuscript. They must report these values in the text or in a table.

Lines 334-335: In my opinion it is better to add a comma between “pH” and “as” and between “soil” and “is uncertain”. This would make the sentence more understandable.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

thank you for including the answers and revised version of the article.

I think this work is now suitable for publication (after revision).

Author Response

Query: Dear Authors, thank you for including the answers and revised version of the article. I think this work is now suitable for publication (after revision).

Response: The author(s) thank the reviewer for his constructive criticism rendered during the first review process. Thank you for accepting our paper in its current revised form. The author(s) are grateful.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors replied to all comments made but unfortunately they did not perform an ANOVA analysis to determine significant differences. For this reason, the results reported are not publishable because they are not statistically demonstrated. I believe that this paper can be published only if the authors statistically verify the differences they found and commented on.

 

Author Response

Query: The authors replied to all comments made but unfortunately they did not perform an ANOVA analysis to determine significant differences. For this reason, the results reported are not publishable because they are not statistically demonstrated. I believe that this paper can be published only if the authors statistically verify the differences they found and commented on.

Response: The main goal of this paper is to evaluate the effectiveness of EICP in immobilizing heavy metals. Our conclusions are based on the experimental results obtained. We do agree that this is a first step in showing the efficacy of the method and additional testing is required to further prove the efficacy of the method. Statistical analysis is clearly beyond the scope of this paper. This paper focusses on the experimental procedures and the corresponding results. It would be feasible to perform a meaningful statistical analysis only after degenerating a larger database that includes several more soil types including a set of other chelants. The data we have in this paper does not qualify for a thorough statistical analysis owing to independent variables. Hence we regret to say that this comment cannot be addressed to the reviewers’ satisfaction at this time and strongly believe that it would be unfair to expect such analysis in this paper. Clearly the other two reviewers see value in publishing the paper as is without any statistical analysis.

Moreover, when we conduct ANOVA analysis, we are attempting to determine if there is a statistically significant difference among the groups (i.e., different parameters). If there is a difference, then we may have to examine where the group differences lay. But in this article, ANOVA analysis seems to be out of scope because we need to perform N - way ANOVA analysis due to more than two independent variables (ES1, ES2 and ES3 are independent variables; ethylene diamine tetra-acetic acid (EDTA) and citric acid (C₆H₈O₇) extractants are totally independent). Due to this, the article may lose its coherence and even continuity of thought.

Author Response File: Author Response.pdf