Microbial Indicators Show the Rehabilitation Flow of Soil Microbiota After the Brumadinho Dam Collapse
, Lutecia Rigueira Medina 1, Alan Emanuel Silva Cerqueira 2, Alex Castro Gazolla 1, Wiane Meloni Silva 1, Igor Rodrigues de Assis 3, Diego Aniceto 4, Sergio Oliveira de Paula 5 and Cynthia Canêdo da Silva 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study tried to monitor the rehabilitation of iron ore tailings-affected soils by using several microbial indicators including metabolic and enzymatic activities and bacterial community structures. Although the microbial indicators adopted in this study may be useful, there are many uncertainness and scientific flaws, bringing bout serious damage to the current manuscript. This manuscript should be improved by reconsidering the structures of backgrounds, methodology, results, and discussion sections.
Specific comments:
L57-73. These descriptions are redundant and lack concreteness. Authors need to provide concrete explanations on microbial indicators for monitoring environmental changes. The concrete descriptions seem to start from L74, so that authors may reconsider the structure of these paragraphs.
L117-123. In Figure 1, where were the 15 and 6 sampling points? Specify these.
L128-136. Methodology is unclear. Please explain how to measure MBR, MBC, and enzyme activities.
L143-157. Do authors need to state appropriate references for the method for analyzing microbial communities?
L167. Specify the items analyzed in this study and clear the methodology.
L172. How did authors measure qCO2? State it in the Methods section.
L179-182, Figure 3. In both Results and Discussion sections, authors need to mention about the seasonal variations of qCO2, besides MBR and MBC. Authors should discuss about why the results of qCO2 were different from those of MBR and MBC.
L193-195, Figure 4. Authors argue the seasonal variation for acid and alkaline phosphatases activities, based on the significant differences only in March samples. However, the appearance of activities in reference and affected areas were clearly different in March 2022 and March 2023. While lower activities were observed in reference area for 2022, those were observed in affected area for 2023. Therefore, the authors’ contention on the seasonal variation is questionable.
L193-195. How about the seasonal variations of other enzyme activities? Authors need to mention about all the results indicated in Figures.
L196-202. While two phosphatases in March 2022 were higher in affected area, other enzymes tended to be higher in reference area (Figure 4). Authors need to state the differences of appearance of these enzyme activities in Results and Discussion sections.
Figure 5. Please explain the plots of orange square and green circle in the Figure legend.
Figure 5, L206-211. In this study, authors argue the importance of microbial indicators such as metabolic and enzymatic activities. Based on only chemical and physicochemical measurements, was it difficult to show the similarity between the affected and reference samples? Readers of this article would want to know about if these microbial indicators significantly affect the judgement for soil rehabilitation obtained by chemical and physicochemical measurements.
Figure 6. Do upper, right columns represent the results from March 2023?
Figure 7, L241-246. How about the fate of these indicator ASVs during the term of March 2022 to March 2023? Authors state the gradual reduction in differences in microbiota over time (L221-222).
L256-278. Most descriptions do not discuss about the results of this study but state the general background of this study. It seems appropriate to remove these to the Introduction section.
L279-285. Authors argue the effect of rainfall in 2022 for the plausible interpretation of microbial indicators. However, this is only a speculation. Authors need to show scientific basis.
L285-288. Please state the reason to consider so.
L290-298. These descriptions also appear to be general discussion. Authors need to discuss specifically on the results obtained in this study.
L345-350. See the comment on L279-285 (above).
L372-385. The result of this study demonstrates the presence of Chloroflexi-related microorganism but not the physiological properties including photosynthetic activity and inorganic assimilation. Therefore, these are over-discussed.
L406-422. The genera observed for the samples should be stated in the Results section.
Author Response
Reviewer 1
Specific comments:
Q1. L57-73. These descriptions are redundant and lack concreteness. Authors need to provide concrete explanations on microbial indicators for monitoring environmental changes. The concrete descriptions seem to start from L74, so that authors may reconsider the structure of these paragraphs.
Answer: The paragraphs have been rewritten to make them clearer. Lines 58 to 74.
Q2. L117-123. In Figure 1, where were the 15 and 6 sampling points? Specify these.
Answer: In total there were 21 points, 15 from the affected area and 6 from the reference (lines 111 to 120)
Q3. L128-136. Methodology is unclear. Please explain how to measure MBR, MBC, and enzyme activities.
Answer: This explanation was added, lines 127 to 199.
Q4. L143-157. Do authors need to state appropriate references for the method for analyzing microbial communities?
Answer: The references were added in the itens 2.3 and 2.4.
Q5. L167. Specify the items analyzed in this study and clear the methodology.
Answer:The methodology section was restructured to explain all the methods employed, lines 127 to 199.
Q6. L172. How did authors measure qCO2? State it in the Methods section.
Answer: The methodology was added in the item 2.2, line 183.
Q7. L179-182, Figure 3. In both Results and Discussion sections, authors need to mention about the seasonal variations of qCO2, besides MBR and MBC. Authors should discuss about why the results of qCO2 were different from those of MBR and MBC.
Answer: The seasonal variations of qCO2 were added in the Results (lines 253 to 255) and Discussion (lines 360 to 373).
Q8. L193-195, Figure 4. Authors argue the seasonal variation for acid and alkaline phosphatases activities, based on the significant differences only in March samples. However, the appearance of activities in reference and affected areas were clearly different in March 2022 and March 2023. While lower activities were observed in reference area for 2022, those were observed in affected area for 2023. Therefore, the authors’ contention on the seasonal variation is questionable.
Answer: The results were rewriting in the lines 266 to 268.
Q9. L193-195. How about the seasonal variations of other enzyme activities? Authors need to mention about all the results indicated in Figures.
Answer: Improved information in the discussion section ( lines 382 to 407).
Q10. L196-202. While two phosphatases in March 2022 were higher in affected area, other enzymes tended to be higher in reference area (Figure 4). Authors need to state the differences of appearance of these enzyme activities in Results and Discussion sections.
Answer: The enzyme activities was rewrited in the Results (lines 269 to 283) and Discussion section (lines 380 to 405).
Q11. Figure 5. Please explain the plots of orange square and green circle in the Figure legend.
Answer: The figure 5 was edited and there is a legend explaining the orange squares and green circles.
Q12. Figure 5, L206-211. In this study, authors argue the importance of microbial indicators such as metabolic and enzymatic activities. Based on only chemical and physicochemical measurements, was it difficult to show the similarity between the affected and reference samples? Readers of this article would want to know about if these microbial indicators significantly affect the judgement for soil rehabilitation obtained by chemical and physicochemical measurements.
Answer: We did not evaluate or compare the importance of biological parameters relative to physico-chemical parameters in assessing and monitoring the soil quality during rehabilitation or recovery efforts. Our study demonstrates that both are crucial for tracking a rehabilitation program (Lima et al., 2024; https://doi.org/10.1016/j.jenvman.2024.120559). However, what we have noticed in our work, data not yet published, is that the response of bioindicators through some bioengineering action are detected more quickly than the responses of physical-chemical parameters, showing the sensitivity of these bioindicators to environmental changes. However, in this work this issue was not evaluated and explanation about the sensitivity of bioindicators was added in the Results, lines 269 to 283.
Q13. Figure 6. Do upper, right columns represent the results from March 2023?
Answer: The figure was updated for a better understanding.
Q14. Figure 7, L241-246. How about the fate of these indicator ASVs during the term of March 2022 to March 2023? Authors state the gradual reduction in differences in microbiota over time (L221-222).
Answer: Information specifying the increased similarity of ASVs over time, lines 310 to 321.
Q15. L256-278. Most descriptions do not discuss about the results of this study but state the general background of this study. It seems appropriate to remove these to the Introduction section.
Answer: Based on the number of lines L256-278, this issue was considered in the discussion, so we agree and this information has been rewritten more objectively, lines 330 to 345.
Q16. L279-285. Authors argue the effect of rainfall in 2022 for the plausible interpretation of microbial indicators. However, this is only a speculation. Authors need to show scientific basis.
Answer: This discussion was improved using scientific information from National Institute of Meteorology (INMET), lines 356-357.
Q17. L285-288. Please state the reason to consider so.
Answer: We agree, this information was removed of text.
Q18. L290-298. These descriptions also appear to be general discussion. Authors need to discuss specifically on the results obtained in this study.
Answer: This section has been rewritten to make the discussion relevant to the context of this study, lines 349-359.
Q19. L345-350. See the comment on L279-285 (above).
Answer: This section has been rewritten to make the discussion relevant to the context of this study, lines 420 to 426.
Q20- L372-385. The result of this study demonstrates the presence of Chloroflexi-related microorganism but not the physiological properties including photosynthetic activity and inorganic assimilation. Therefore, these are over-discussed.
Answer: This section has been rewritten, lines 448 to 461.
Q21- L406-422. The genera were indicated for the samples should be stated in the Results section.
Answer: These genera were showed in Results section cited in the figure 6.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis research aimed to evaluate various microbial indicators in the soil, such as enzyme activity, microbial biomass carbon, microbial basal respiration, and microbial diversity, to monitor the rehabilitation of the affected area. The study provides valuable insights into the use of microbial indicators for monitoring soil recovery in mining-affected areas, highlighting the potential for using microbial indicators to monitor soil quality and ecosystem services in areas impacted by mining activities.
However, there are still many key issues in the article that have not been resolved. It is recommended to address these issues before publication.
1. The authors have compared microbial indicators across different time periods in the mining area soil. Although the discussion section mentions that microbial community indicators may be influenced by factors such as rainfall and seasons (Page 5, Lines 180-182), there is no introduction to the relevant conditions of rainfall and climate environment in the study area, which is crucial for the discussion section. It is recommended to supplement this information.
2. The discussion also suggests that changes in microbial indicators may be due to vegetation restoration (Page 10, Lines 257-261), but there is no such information in the background introduction of the mining area. It is recommended to clearly state the significance of comparing soil biotic indicators across different time periods.
3. Sections and paragraphs: Please pay attention to the division of sections and paragraphs, particularly the methods and results sections, which are quite dense.
4. Strengths and Limitations: The conclusion section is suggested to include more information on the potential limitations of the techniques used, such as the effect of sensitivity and specificity of the microbial indicators selected.
5. Data Presentation: some of the data could be better visualized to highlight trends and differences more effectively. For example, the use of more comparative graphs or heatmaps for microbial diversity data could enhance the presentation.
Author Response
Reviewer 2
Q1. The authors have compared microbial indicators across different time periods in the mining area soil. Although the discussion section mentions that microbial community indicators may be influenced by factors such as rainfall and seasons (Page 5, Lines 180-182), there is no introduction to the relevant conditions of rainfall and climate environment in the study area, which is crucial for the discussion section. It is recommended to supplement this information.
Answer: This information was added throughout the discussion lines 354 to 357.
Q2. The discussion also suggests that changes in microbial indicators may be due to vegetation restoration (Page 10, Lines 257-261), but there is no such information in the background introduction of the mining area. It is recommended to clearly state the significance of comparing soil biotic indicators across different time periods.
Answer: This section has been rewritten ( lines 330 to 345). However, there is no history of the area in question before it was affected by the iron ore tailings, our studies in the area began shortly after the removal of this tailings.
Q3. Sections and paragraphs: Please pay attention to the division of sections and paragraphs, particularly the methods and results sections, which are quite dense
Answer: These issues were fixed.
Q4. Strengths and Limitations: The conclusion section is suggested to include more information on the potential limitations of the techniques used, such as the effect of sensitivity and specificity of the microbial indicators selected.
Answer: This information was added in the Conclusions section (lines 532 to 537).
Q5. Data Presentation: some of the data could be better visualized to highlight trends and differences more effectively. For example, the use of more comparative graphs or heatmaps for microbial diversity data could enhance the presentation.
Answer: The microbial diversity figures have been optimized to clearly present the results, Figure 6.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors1. The abstract claims significant findings, but the lack of numerical data or statistical outcomes (e.g., makes the results less impactful.
2. The introduction extensively discusses microbial indicators but overlooks their specific relevance to the context of iron ore tailings. A stronger link to the study area and its unique challenges would improve relevance.
3. Seasonal variations are discussed in the results but are not adequately accounted for in the methodology. Could you explain how these variations were controlled or considered during sample collection and analysis?
4. Statistical methods are mentioned but not described in detail.
5. While recovery trends are noted, the discussion does not critically address whether these trends are statistically significant or biologically meaningful.
6. Although microbial diversity is mentioned as an indicator, the results do not thoroughly integrate metataxonomic findings. For example, which microbial taxa were most responsive to rehabilitation, and what ecological roles do they play?
7. The discussion section is sometimes repetitive and does not fully contextualize findings within the broader literature. For example, comparisons with similar studies on post-mining rehabilitation are sparse.
8. The conclusions are general and do not emphasize the key takeaways or implications of the study.
Comments on the Quality of English Languageminor revision required
Author Response
Reviewer 3
Q1- The abstract claims significant findings, but the lack of numerical data or statistical outcomes (e.g., makes the results less impactful.
Answer: The abstract section has been rewritten, lines 26 to 53.
Q2. The introduction extensively discusses microbial indicators but overlooks their specific relevance to the context of iron ore tailings. A stronger link to the study area and its unique challenges would improve relevance.
Answer: Scientific informations from other mining area studies was added to the discussion section such as lines 413 to 415; 444 to 447.
Q3. Seasonal variations are discussed in the results but are not adequately accounted for in the methodology. Could you explain how these variations were controlled or considered during sample collection and analysis?
Answer: The meteorological data used in this study were consulted at the National Institute of Meteorology (INMET, https://portal.inmet.gov.br/dadoshistoricos) (lines 120 to 122)
Q4. Statistical methods are mentioned but not described in detail.
Answer: The 2.4 section has been rewritten.
Q5. While recovery trends are noted, the discussion does not critically address whether these trends are statistically significant or biologically meaningful.
Answer: The section has been rewritten to be clearer (lines 448 to 449).
Q6. Although microbial diversity is mentioned as an indicator, the results do not thoroughly integrate metataxonomic findings. For example, which microbial taxa were most responsive to rehabilitation, and what ecological roles do they play?
Answer: The section has been rewritten to be clearer (lines 482 to 489).
Q7. The discussion section is sometimes repetitive and does not fully contextualize findings within the broader literature. For example, comparisons with similar studies on post-mining rehabilitation are sparse.
Answer: The discussion section has been restructured, and was added references from studies of mining areas such as lines 413 to 415; 444 to 447.
Q8. The conclusions are general and do not emphasize the key takeaways or implications of the study.
Answer: The Conclusions section has been restructured.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsDear Sir,
The manuscript is interesting, but lacks in some pertinent comparisons:
- are there any data of soil biodiversity before the collapse of the dam? (either in the same area, or in similar areas)
- iron mining soils in Brazil have been studied previously – e.g. Silva et al., Soil microbiological attributes indicate recovery of an iron mining area and of the biological quality of adjacent phytophysiognomies, Ecological Indicators, 2018, 93, 42-151; perhaps a comparison with such studies could be performed as well.
- with other similar studies of soil characteristics after a dam collapse – e.g. Couto et al., Physical, chemical and microbiological characterization of the soils contaminated by iron ore tailing mud after Fundão Dam disaster in Brazil, Applied Soil Ecology, 2021, 158, art.no. 103811 (a Couto et al. paper was cited in the text, but is not present in the list of references)
- were natural seasonal variations taken into account? In fig. 6, left hand bottom part – Reference area: shouldn’t march 2022 be similar to march 2023, since this area was not affected?
Other comments:
- literature citation in the text: use either lowercase or uppercase throughout the entire manuscript, but not both;
- the authors mention 15 collection point + 6 reference points; Fig. 1 presents 23 points (thus, +2 points). It would be good to mention in this figure which are a investigation points and which are the reference points (maybe by highlighting them in different colors?).
- pits were dug at depth of “0-20 cm” – does this means that surface soil was also collected
- “To characterize the microbial diversity of the soil, 10-point representatives of the AA 138
and REF groups were sampled. Four samples (Pt3, Pt7, Pt12, and Pt17) belong to the Af- 139
fected Area group, while six (Pt9, Pt10, Pt14, Pt15, Pt19, and Pt20) were from the Reference 140
area.” Why chose 15 collection points, if only 4 were effectively used? The other samples were used only for physico-chemical analyses
- is fig.3 necessary, since it presents the same data as fig 2? And there are some typos in this figure – march 2023 appears 2 times
- fig. 6 left hand bottom part – Reference area – 1,2 and 3 are presented as “Affected area”
- fig. 7: could the genus be presented from the highest to the lowest?
Otherwise, the manuscript is well-written and presents interesting data. It could be accepted after addressing the comments above.
Author Response
Reviewer 4
Q1- Are there any data of soil biodiversity before the collapse of the dam? (either in the same area, or in similar areas)
Answer: Unfortunately, there is no history of soil biodiversity from affected areabefore the collapse of the dam.
Q2- Iron mining soils in Brazil have been studied previously – e.g. Silva et al., Soil microbiological attributes indicate recovery of an iron mining area and of the biological quality of adjacent phytophysiognomies, Ecological Indicators, 2018, 93, 42-151; perhaps a comparison with such studies could be performed as well.
Answer: Other studies on post-mining soil rehabilitation were incorporated into the Discussion, such as lines 413 to 415; 444 to 447. It is worth noting that comparing our data with other studies is a difficult task due to the different bioengineering actions applied in each affected area. For example, in the area affected by the Fundão dam in Mariana, the tailings were not removed, whereas in the area of ​​this study, the tailings were completely removed as the first rehabilitation action of the area.
Q3- With other similar studies of soil characteristics after a dam collapse – e.g. Couto et al., Physical, chemical and microbiological characterization of the soils contaminated by iron ore tailing mud after Fundão Dam disaster in Brazil, Applied Soil Ecology, 2021, 158, art.no. 103811 (a Couto et al. paper was cited in the text, but is not present in the list of references)
Answer: The reference was removed of the text.
Q4- Were natural seasonal variations taken into account? In fig. 6, left hand bottom part – Reference area: shouldn’t march 2022 be similar to march 2023, since this area was not affected?
Answer: The seasonal variations were considered for data analisys. The discussion of the differences between March 2022 and March 2023 was incorporated (lines 356 to 359; 420 to 423).
Other comments:
Q5- literature citation in the text: use either lowercase or uppercase throughout the entire manuscript, but not both;
Answer: This issue was fixed.
Q6- the authors mention 15 collection point + 6 reference points; Fig. 1 presents 23 points (thus, +2 points). It would be good to mention in this figure which are a investigation points and which are the reference points (maybe by highlighting them in different colors?).
Answer: We were unable to change the colors of the points on the map, but we made this information clearer in the text, lines 111 to 120.
Q7- pits were dug at depth of “0-20 cm” – does this means that surface soil was also collected
Answer: The soil samples from 0-20 cm was collected, including the surface soil, line 116.
Q8- “To characterize the microbial diversity of the soil, 10-point representatives of the AA and REF groups were sampled. Four samples (Pt3, Pt7, Pt12, and Pt17) belong to the Affected Area group, while six (Pt9, Pt10, Pt14, Pt15, Pt19, and Pt20) were from the Reference area.” Why chose 15 collection points, if only 4 were effectively used? The other samples were used only for physico-chemical analyses
Answer: This issue was rewritten to be be clearer. In total there were 21 points, 15 from the affected area and 6 from the reference, lines 111 to 120.
Q9- Is fig.3 necessary, since it presents the same data as fig 2? And there are some typos in this figure – march 2023 appears 2 times
Answer: We moved figure 2 for supplementary material and kept figure 3 (now named figure 2) in the main manuscript.
Q10- fig. 6 left hand bottom part – Reference area – 1,2 and 3 are presented as “Affected area”
Answer: The figure was improved for a better understanding.
Q11- fig. 7: could the genus be presented from the highest to the lowest?
Answer: The figure was improved following the advice.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has been revised extensively according to the reviewer's comments and it is acceptable.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have signficantly improved this paper, and it could be accepted now.
Reviewer 4 Report
Comments and Suggestions for AuthorsDear Sir,
The authors have satisfactorily answered to all queries (except Fig. 7, where the genus are still randomly presented). The manuscript can now be accepted for publication.
