Cadmium-Tolerant Bacteria in Cacao Farms from Antioquia, Colombia: Isolation, Characterization and Potential Use to Mitigate Cadmium Contamination

Round 1

Reviewer 1 Report

 

The paper by Quiroga-Mateus et al. addresses an interesting topic in bioremediation of cadmium in cacao growing farms in Colombia. The study question is important, as for the cocoa producers as well as for the consumers. In the study the authors managed to isolate five cadmium-tolerant Bacillus strains from the studied plantations. Furthermore, they demonstrated that bioaugmentation with cadmium-tolerant bacteria with addition of zeolite has the superior capability for Cd removal in field conditions.

Unfortunately, although the topic is interesting and the experimental design is appropriate, the paper is poorly written and has some serious flaws in data analysis and presentation. Here I will draw attention to the biggest issues, and the rest of the corrections and comments are in the attached pdf file.

-     - The quality of English is quite poor, but also often there are missing parts of the sentences which makes it difficult to read and understand the meaning.

-      -  At many instances the references in the text to a table or figure are not appropriate and the reader gets confused and mislead.

-   - In the Introduction the aims of the study should be stated more clearly, point by point. Also, since a lot of attention thorough the paper, and especially in the discussion, is given to assessing the applicability of IMC method for measuring the Cd immobilization activity, it should be stated as one of the aims of the study

-       - My biggest concern is the lack of appropriate statistical testing, stating the number of replicates used and reporting the variability of measured parameters. Without these data I cannot assess the scientific soundness of the obtained results.

-      - The authors have collected fresh cacao seeds and measured the content of Cd in them, but the results are not presented nor discussed

-     - Discussion is another weak point of this paper. It is not streamlined and well connected with their results. The authors do not discuss their obtained results in much depth. The papers that are referenced are predominantly their previous studies. If there is not much literature on Cd remediation from cacao plantations, I am sure that the discussion could be expanded to remediation of Cd in different types of crops, or even the remediation of other pollutants in cacao plantations. Thus, the discussion needs much improvement.

Comments for author File: Comments.pdf

Author Response

1. Authors would like to thank the reviewer for the valuable comments throughout the manuscript. They helped to reshape the document in a much positive manner.
2. Authors agree with the reviewer regarding the quality of English and the incomplete sentences which makes it difficult to read and understand the meaning.

Answer: Authors asked an English-speaking person for the final proofreading. It was performed throughout the manuscript.

3. At many instances the references in the text to a table or figure are not appropriate and the reader gets confused and mislead.

Answer: Agree, the references in the text of the tables and figures were reviewed and adjusted if necessary. We identified the error citing Figure 3 but at the same time the major results were not explained. Instead, authors discussed Figure 2. The text was adjusted accordingly. Also, the legends of Table 3 (formerly Tables 2 & 4) and 4 were adjusted for better understanding.

4. In the Introduction the aims of the study should be stated more clearly, point by point. Also, since a lot of attention thorough the paper, and especially in the discussion, is given to assessing the applicability of IMC method for measuring the Cd immobilization activity, it should be stated as one of the aims of the study.

 Answer: Agree, authors included three major aims of this manuscript more clearly, point by point, in lines: 107-113. Besides, the application of IMC method for measuring the Cd immobilization activity was stated in both the Abstract and the Introduction sections, in lines 22-23, 89-90 and 110-111.

5. My biggest concern is the lack of appropriate statistical testing, stating the number of replicates used and reporting the variability of measured parameters. Without these data I cannot assess the scientific soundness of the obtained results.

 Answer: Agree, authors addressed the comment from Reviewer 1. Descriptive statistics were included for table 2 (formerly tables 2 and 4). Moreover, an explanation was made in the discussion section (lines 567-568 and 624-627) regarding the preliminarily of the results obtained on field trials, however, with biological sound about Cd-immobilization.

6. The authors have collected fresh cacao seeds and measured the content of Cd in them, but the results are not presented nor discussed

 Answer: Agree, despite collecting the cacao seeds, they were not chemically analysed due to COVID and other constrains. Therefore, the text mentioning the collection of seed was deleted for clarity of the other parts of the work.

7. Discussion is another weak point of this paper. It is not streamlined and well connected with their results. The authors do not discuss their obtained results in much depth. The papers that are referenced are predominantly their previous studies. If there is not much literature on Cd remediation from cacao plantations, I am sure that the discussion could be expanded to remediation of Cd in different types of crops, or even the remediation of other pollutants in cacao plantations. Thus, the discussion needs much improvement.

 Answer: Agree, authors improved the discussion section according to reviewers’ suggestions and comments. New literature was included to not only base the discussion on authors’ previous publications. In the revised version, the discussion included a comparison between CdtB strains isolated from cacao in Colombia in a previous work. New literature was also included from other groups in bioremediation, see lines 527 and 537; as well as 599-601.

Comment to the reviewer 1: Besides our answer to the above-mentioned comments, the suggestions made by reviewer 1 in the PDF attached in the report were also considered and included in the final version of the manuscript. Some changes in paragraphs were performed. The table in the PDF attached it is explained the line of the change made accordingly.

Author Response File: Author Response.pdf

Reviewer 2 Report

 

This study presents the possibility of Cd bioremediation using bacteria isolated from farm soils. Five strains were used for laboratory assessment of Cd immobilization while two other stains from the previous study were used, combined with zeolite application, for farm trial assessment. Both laboratory and farm trials show promising results of Cd immobilization, which helps develop feasible bioremediation protocols to tackle Cd contamination in cacao tree farms. The manuscript was well written and organized, I recommend considering it for publication in Processes after addressing my concerns.

 

1) The authors may want to explain why strains CdtB13 and CdtB14 isolated from farms A and D soils that show promising immobilization in the laboratory study do not show enhanced Cd immobilization in the farm soils in the farm trial study?

 

2) There are two tables (Tables 2 and 4) of soil properties variations (or Table S3), it will be helpful for readers to understand how these strains work to immobilize Cd if the authors explain or speculate how these properties changes (e.g., decrease or increase of CEC, Ca, Mg, K, P, etc.) result from or into bacterial immobilization activities.

 

3) How do the authors explain the farm-to-farm variations when applying CdtB50 and CdtB70 to farms B and C, respectively? What if the same strain was applied to different plots on the same farm?

 

4) The authors may want to compare the growth and immobilization activity of the five strains isolated in the current study with those isolated in the previous study to have a better understanding of if the current strains will be promising to immobilize Cd in farm soils.

 

5) I would have expected that the authors discuss how these bacterial strains immobilize Cd, e.g., secreting extracellular substances and chelating Cd or absorbing Cd and complexing Cd in vivo? This discussion will help understand how stable the immobilization is.

 

5) L106, 110: “by [15]”, “by [16]”. L141, in [23]. Change to “by Author [citation #]” and in Author [#].

 

6) L139, change “he” to “the”.

 

7) L155-158, “50 mM 6 mL L–1”, “80 mM 5 mL L–1”, and “20 mM 2 mL L–1” … are confusing. Correct them to # mL ## mM if they stand for a volume of solution with a concentration of solutes.

 

8) L175, add “.” after [11].

 

9) like comment 7, L238, “mL L–1” is neither a concentration unit nor a volume unit. Same issue in L249 “μL L–1”.

 

10) Table 2, combine with Table 4 to show a good comparison before and after bacteria application, then Table S1 is not needed (it is not cited in the text yet though).

 

11) Number all sub-headings like “2.1. Soil samples origin …”.

 

Author Response

Authors would like to thank reviewer 2 for the constructive comments and suggestion and the positive review made to our work.

1. The authors may want to explain why strains CdtB13 and CdtB14 isolated from farms A and D soils that show promising immobilization in the laboratory study do not show enhanced Cd immobilization in the farm soils in the farm trial study?

 Answer: Even though, the strains CdtB13 and 14 have shown great Cd immobilization capacities in the laboratory conditions, they are in the initial stage of characterization through the bioprospection process. On the contrary, the strains A and B (the nomenclature of the strains was changed since it has Intelectual property by the bioproduct is still in development by Agrosavia), used in the field trial are in an advanced status of bioproduct development, another process, currently in pre-formulated stage. This is important to point out, since there is no sense to apply single bacterial inoculum in the very first step of the process, without any conditioning to the environmental stress that the strains will face in the field conditions. It is necessary the bioproduct development process. This explanation was included within the discussion section, in lines 557-566.

2. There are two tables (Tables 2 and 4) of soil properties variations (or Table S3), it will be helpful for readers to understand how these strains work to immobilize Cd if the authors explain or speculate how these properties changes (e.g., decrease or increase of CEC, Ca, Mg, K, P, etc.) result from or into bacterial immobilization activities.

 Answer: Agree, authors did not observe a direct influence of CdtB into the soil properties changes. However, we describe the possible effect the CdtB have in Cd immobilization during the field trial. This was pointed out in the discussion section, in lines: 584-585. The effect of CdtB in some parameters was included in lines 571-589.

3. How do the authors explain the farm-to-farm variations when applying CdtB50 and CdtB70 to farms B and C, respectively? What if the same strain was applied to different plots on the same farm?

 Answer: Agree, authors included a better explanation of the farms, distances between them and size. Due to the latest, the experiment could not be performed in the common randomized block in the same field, i.e., same strain in different plots of the same farm. In previous research conducted in Ecuador (Arguello, et al., 2019), the average coefficient of variation within same field (farm size <2 ha) is nearly 40% and farms among the same canton about 50%. Authors expect the same variation in our fields.

4. The authors may want to compare the growth and immobilization activity of the five strains isolated in the current study with those isolated in the previous study to have a better understanding of if the current strains will be promising to immobilize Cd in farm soils.

 Answer: Agree. The immobilization capacity (Joules or ‘J’ of maximum heat produced Q_max in the calorimetric thermograms) and the immobilization ratios (mg L^-1 of Cd in liquid media in the immobilization test) were included in the discussion section. The growth rate was not considered in the initial version of the manuscript. In this new version, the maximum growth rate (µ_max) and the adaptation phase (λ) of the new strains were compared with the positive control strains, as well as, three strains isolated in a previous bioprospection (Bravo et al., 2018). This was included in lines 504-515.

5. I would have expected that the authors discuss how these bacterial strains immobilize Cd, e.g., secreting extracellular substances and chelating Cd or absorbing Cd and complexing Cd in vivo? This discussion will help understand how stable the immobilization is.

 Answer: Agree, the experimental set up was not meant for determining the exact mechanism of the CdtB immobilization strategy. However, authors included some discussion in lines 519-532 and suggested that due to P and K solubilization observed in lab (data not shown), the metabolic pathway could be bioweathering and biochelation. These mechanisms should be tested further.

 L106, 110: “by [15]”, “by [16]”. L141, in [23]. Change to “by Author [citation #]” and in Author [#].

Answer: Edited accordingly through the whole manuscript.

7. L139, change “he” to “the”.

Answer: Edited accordingly. Spell check.

8. L155-158, “50 mM 6 mL L–1”, “80 mM 5 mL L–1”, and “20 mM 2 mL L–1” … are confusing. Correct them to # mL ## mM if they stand for a volume of solution with a concentration of solutes.

Answer: Edited accordingly. All the concentrations were showed in mass/volume or mass/mass relations.

9. L175, add “.” after [11].

Answer: Edited accordingly.

10. like comment 7, L238, “mL L–1” is neither a concentration unit nor a volume unit. Same issue in L249 “μL L–1”.

Answer: Edited accordingly.

11. Table 2, combine with Table 4 to show a good comparison before and after bacteria application, then Table S1 is not needed (it is not cited in the text yet though).

 Answer: Agree, the tables were combined in a single table now Table 3 for better understanding of the data. Line 441.

12. Number all sub-headings like “2.1. Soil samples origin …”.

Answer: The number of all sub-headings were included accordingly.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear editor and authors:

       This article focused on the isolation of cadmium-tolerant bacteria in cacao-growing farms to bioremediate heavy metal (Cd²⁺, etc.) contaminated soils. The results showed that one of the isolated cadmium-tolerant bacteria (CdtB14) had advantages in growth and heat ratios and could reduce the titer of Cd²⁺ in the cacao-growing farms. This work is probably useful to improve the application of cadmium-tolerant bacteria in bioremediation of soil farmlands. However, several serious issues should be addressed before the acceptance of this manuscript.

1.      Besides growth and heat ratios, other metabolic activities of CdtB should be analyzed by their genomes.

2.      The strong Cd²⁺ immobilization capacity by CdtB14 should be explained by difference analysis with other isolated strains.

3.      Please explain the synergistic effect of different CdtB on Cd²⁺ immobilization.

4.      The effect of cadmium-tolerant bacteria on the other components in the soils must be evaluated.

5.      Ca⁺² and Mg⁺² should be “Ca²⁺”, “Mg²⁺”.

6.      The errors should be supplemented in all tables.

7.      No space should be added between temperature and centigrade.

Author Response

1. Besides growth and heat ratios, other metabolic activities of CdtB should be analyzed by their genomes.

Answer: Authors agree on the necessity for including metabolic analysis in these studies, however, due to the lack of funding and the pandemic this part was not included in our study. However, in other parallel study in Antioquia (different farm’s placed) a comparison between the CdtB community from soil subsamples taken when the maximum peak of heat-flow was detected by calorimetry. The paper is in the writing process and will be submitted for publication later this year. Authors included some discussion in lines 547-555.

2. The strong Cd²⁺ immobilization capacity by CdtB14 should be explained by difference analysis with other isolated strains.

Answer: Agree, this was explained in a better way in the discussion section (lines 495-498. A paragraph comparing some maximal Cd immobilization rates and capacities to other CdtB related to crops besides cacao has been included Line 591-603.

3. Please explain the synergistic effect of different CdtB on Cd²⁺

Answer: The synergistic effect could be addressed once the CdtB are in a consortium as active biomolecules in a final bioproduct. At this point the CdtB strains isolated in this study were analysed in independent Mergeay medium. The synergistic effect and the side effects of application of the consortium should also be the target in forthcoming studies but we have to wait for the development of the bioproduct, e.g., formulation etc. A brief text was included in the discussion section in lines 574-580.

4. The effect of cadmium-tolerant bacteria on the other components in the soils must be evaluated.

Answer: According to the reviewer 2, this was adjusted, in lines 571-589.

5. Ca⁺² and Mg⁺² should be “Ca²⁺”, “Mg²⁺”.

Answer: This edition was made accordingly.

6. The errors should be supplemented in all tables.

Answer: This was edited accordingly.

7. No space should be added between temperature and centigrade.

Answer: the change was made accordingly in lines: 161, 165, 182-183, 208, 223, 253

Again, authors thank all reviewers for all the comments and suggestions made by the three reviewers and expect that our answers have addressed all.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised paper by Quiroga-Mateus et al. is much improved and now of much higher quality. English corrections and proofreading have made it much more understandable. Furthermore, the majority of my questions and concerns have been addressed, explained and corrected. The new version of discussion is now of satisfactory quality.

Here are my remaining major comments:

1.       Experiments in sections 2.4 and 2.5 use different positive controls (the positive control used in 2.5 should be stated in that section). Please clarify why the use of completely different bacterial species.

2.       Results presented in Table 2 and Figure 3 should be statistically tested. There is enough data to conduct ANOVA and post hoc tests to verify the differences between tested strains. The statistical tests used should be included in section 2.8.

3.       Table 4 is redundant. It shows only the differences between “before” and “after” measurements presented in Table 3. If you still want to present these data, you can add it as an additional row called “change” or alike, for each farm in Table 3.

The minor corrections are highlighted in the comments of the attached pdf document.

Comments for author File: Comments.pdf

Author Response

Here are my remaining major comments:

 

1. Experiments in sections 2.4 and 2.5 use different positive controls (the positive control used in 2.5 should be stated in that section). Please clarify why the use of completely different bacterial species.

Answer: The explanation about why we use different positive controls was included in methods section, in lines 196 – 197, 225, 227 – 228.

 

2. Results presented in Table 2 and Figure 3 should be statistically tested. There is enough data to conduct ANOVA and post hoc tests to verify the differences between tested strains. The statistical tests used should be included in section 2.8.

 

Answer: The explanation about the statistical tests was included in methods section 2.8, in lines 303 - 305. The significance value of the data was included in the lines 364.

 

3. Table 4 is redundant. It shows only the differences between “before” and “after” measurements presented in Table 3. If you still want to present these data, you can add it as an additional row called “change” or alike, for each farm in Table 3.

 

Answer: Table 4 was deleted since reviewers 1 and 2 made the same suggestion.

 

The minor corrections were highlighted in the comments of the attached pdf document.

 

Answer: We appreciate the comments in the attached PDF from reviewer 1. The table in the PDF attached explain the lines of the manuscript where we made the suggested edition.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript has been improved after addressing all comments accordingly and could be published after correcting the following errors.

 

1) Table 4 is not cited in the manuscript, cite it or delete it as it delivers the same information as Table 3. Cite Table S1 in the main text.

 

2) Recommend the authors check the format of the manuscript. For example, L112: "A, B, C and D" should be "A, B, C, and D"; L121: [19] is in bold; L574: bi^ochelation [8]. ...

 

Author Response

Comments and Suggestions for Authors: The manuscript has been improved after addressing all comments accordingly and could be published after correcting the following errors.

1. Table 4 is not cited in the manuscript, cite it or delete it as it delivers the same information as Table 3. Cite Table S1 in the main text.

Answer: Table 4 was deleted and Table S1 was cite in the line 397

2. Recommend the authors check the format of the manuscript. For example, L112: "A, B, C and D" should be "A, B, C, and D"; L121: [19] is in bold; L574: bi^ochelation [8]. ....

Answer: The edition was done accordingly in lines 116, 118, 121 and 588.

Author Response File: Author Response.pdf

Reviewer 3 Report

Several problems previously mentioned still are not sovled and the results should be deeply discussed.

Author Response

Comments and Suggestions for Authors: Several problems previously mentioned still are not sovled and the results should be deeply discussed.

Answer: Authors regret that reviewer 3 is not satisfied with the modifications made in the revised manuscript. Authors partially agree with the comments and suggestions made by reviewer 3 in the first round of the peer review process. The comments regarding genomic analysis and additional assessment for immobilization capacity of the CdtB strains are desired in a research study as the one we are presenting; however, these analyses are, in our opinion, complimentary and depend upon the objective of the study itself. Again, authors appreciate the excellent comments and suggestions made but the reviewer and regret that some of those could not be possible to make.

Additionally, authors would like to reinforce that some other comments were made to the revised version. Those comment (from the different reviewers) were done to improve the discussion section, particularly. Bellow the list of comments and suggestions made by reviewer 3 and the response prepared by authors in round 1 of the peer-review process.

 Our responses in previous round 1:

Dear editor and authors: This article focused on the isolation of cadmium-tolerant bacteria in cacao-growing farms to bioremediate heavy metal (Cd²⁺, etc.) contaminated soils. The results showed that one of the isolated cadmium-tolerant bacteria (CdtB14) had advantages in growth and heat ratios and could reduce the titer of Cd²⁺ in the cacao-growing farms. This work is probably useful to improve the application of cadmium-tolerant bacteria in bioremediation of soil farmlands. However, several serious issues should be addressed before the acceptance of this manuscript.

Authors would like to thank reviewer 3 for the valuable comments and suggestions which allowed to improve the quality of the manuscript.

1. Besides growth and heat ratios, other metabolic activities of CdtB should be analyzed by their genomes.

Answer: Authors agree on the necessity for including metabolic analysis in these studies, however, due to the lack of funding and the pandemic this part was not included in our study. However, in other parallel study in Antioquia (different farm’s placed) a comparison between the CdtB community from soil subsamples taken when the maximum peak of heat-flow was detected by calorimetry. The paper is in the writing process and will be submitted for publication later this year. Authors included some discussion in lines 547-555.

2. The strong Cd²⁺ immobilization capacity by CdtB14 should be explained by difference analysis with other isolated strains.

Answer: Agree, this was explained in a better way in the discussion section (lines 495-498. A paragraph comparing some maximal Cd immobilization rates and capacities to other CdtB related to crops besides cacao has been included Line 591-603.

3. Please explain the synergistic effect of different CdtB on Cd²⁺

Answer: The synergistic effect could be addressed once the CdtB are in a consortium as active biomolecules in a final bioproduct. At this point the CdtB strains isolated in this study were analysed in independent Mergeay medium. The synergistic effect and the side effects of application of the consortium should also be the target in forthcoming studies but we have to wait for the development of the bioproduct, e.g., formulation etc. A brief text was included in the discussion section in lines 574-580.

4. The effect of cadmium-tolerant bacteria on the other components in the soils must be evaluated.

Answer: According to the reviewer 2, this was adjusted, in lines 571-589.

5. Ca⁺² and Mg⁺² should be “Ca²⁺”, “Mg²⁺”.

Answer: This edition was made accordingly.

6. The errors should be supplemented in all tables.

Answer: This was edited accordingly.

7. No space should be added between temperature and centigrade.

Answer: the change was made accordingly in lines: 161, 165, 182-183, 208, 223, 253

Again, authors thank all reviewers for all the comments and suggestions made by the three reviewers and expect that our answers have addressed all.

Author Response File: Author Response.pdf