Microbial Community Shifts and Functional Constraints of Dechlorinators in a Legacy Pharmaceutical-Contaminated Soil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study is meaningful, the data is detailed and the results could be of certain scientific relevance and of interest for the readers of the journal. Before accepting the manuscript, further modifications and improvements are needed on the following points.

1. Line 64-70 Suggest changing the contents of five lines to short sentences;
2. Line106 “the screening thresholds” should be “screening threshold values”;‌

3.Line120   Add the full name of BTEX abbreviation （Benzene, Toluene, Ethylbenzene, and Xylene）；

4.Line126-127   Suggest changing the contents of two lines to short sentences;

5.Line139-141   Supplement the number of parallel samples for measuring the physical and chemical properties of soil samples；

6. Line 171 (P<0.05) and Line 177 (R² values) The “P” and ”R” should be italicized;

7.Line279-280 Microbial genus level scientific names should be italicized；

8.Line 345  The “p” in Firure 5A should be “P”；

9. Line 510 Improve Figure 8, deepen the color of the connections in the network diagram;

10.Line537-541 The conclusion mentions the need to enhance repair strategies, but does not specify how to optimize them (such as adding electron donors, pH adjustment). Suggest providing targeted recommendations based on the research findings of this study.

 

Author Response

Comments 1: Line 64-70 Suggest changing the contents of five lines to short sentences

Response 1: Thank you for pointing this out. Revised accordingly.

Comments 2: Line 106 “the screening thresholds” should be “screening threshold values

Response 2: Agreed with the point. Revised accordingly.

Comments 3: Line 120 Add the full name of BTEX abbreviation （Benzene, Toluene, Ethylbenzene, and Xylene）

Response 3: Agreed with the point. Therefore, we have added the full abbreviation of BTEX.

Comments 4: Line126-127 Suggest changing the contents of two lines to short sentences

Response 4: Agreed with the point. Revised accordingly.

Comments 5: Line139-141 Supplement the number of parallel samples for measuring the physical and chemical properties of soil samples

Response 5: Agreed with the point. Therefore, we supplement the number of parallel samples for measuring the physical and chemical properties of soil samples.

Comments 6: Line 171 (P<0.05) and Line 177 (R2 values) The “P” and ”R” should be italicized

Response 6: Agreed with the point. Revised accordingly.

Comments 7: Line279-280 Microbial genus level scientific names should be italicized

Response 7: Agreed with the point. Revised accordingly.

Comments 8: Line 345  The “p” in Firure 5A should be “P”

Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we made modifications to Figure 5A.

Comments 9: Line 510 Improve Figure 8, deepen the color of the connections in the network diagram

Response 9: Thank you for pointing this out. We agree with this comment.Therefore, we made modifications to Figure 8.（Line 512）

Comments 10: Line537-541 The conclusion mentions the need to enhance repair strategies, but does not specify how to optimize them (such as adding electron donors, pH adjustment). Suggest providing targeted recommendations based on the research findings of this study

Response 10: Agreed with the point. Added two factors(adding electron donors, pH adjustment).

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting research paper from the topical field of microbial community shifts and function responses in long-term contaminated soil. By analysis of the relationship of microbes and physicochemical properties, the authors found the significant role of soil pH, moisture content, pollutant type, and depth on the microbial community. They further evaluated the abundance of dechlorinating microbes, which presents positive correlation with BTEX-degrading and fermentative taxa. The work contributes valuable knowledge, which helps further develop microbial remediation strategies towards legacy-contaminated sites. This manuscript is well-written and well-organized. I recommend it for publication after minor revisions.

My comments:

1. Too many Keywords.
2. Introduction line 72: I think it is better to insert a short introduction here to show the scale and representativeness of historically contaminated sites with chlorinated organic compounds.
3. Line 134: revise 5 g to “5.0 g”?
4. Line 171: italic of “P”
5. Line 143: The quantitative method and detection limit of pollutants should be provided
6. It is better to add declaration of the symbols such as “*” in the legend.

Author Response

Response to Reviewer 2 Comments

Comments 1: Too many Keywords

Response 1: Thank you for pointing this out. Agreed with the point. Two keywords (BTEX degradation; Soil pollution) have been deleted.

Comments 2: Introduction line 72: I think it is better to insert a short introduction here to show the scale and representativeness of historically contaminated sites with chlorinated organic compounds

Response 2: Agreed with the point.

Comments 3: Line 134: revise 5 g to “5.0 g”?

Response 3: Thank you for pointing this out. We agree with this comment and made the necessary revisions.

Comments 4: Line 171: italic of “P”

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we changed it to italic.

Comments 5: Line 143: The quantitative method and detection limit of pollutants should be provided.

Response 5: Revised accordingly. Added detection method and detection limit.

Comments 6: It is better to add declaration of the symbols such as “*” in the legend.

Response 6: Thank you for pointing this out. We agreed with this comment and added the declaration of “*” in the legend.

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigated the microbial ecological responses and natural attenuation potential at a decommissioned pharmaceutical-chemical site heavily contaminated. These findings provide new insights into microbial ecology under complex organic pollution. However, the authors need to address the following issues in order to make this manuscript suitable for publication in the journal.

1. Improve the abstract in the manuscript in the order of Purpose, Methods, Results, and Conclusions;
2. Suggest reducing the number of keywords;
3. Suggest further analysis of the negative correlation between pH and SMC on the dechlorinators, which may be indirectly affected by the redox potential;
4. Suggest supplementing qPCR detection of dechlorination functional genes, or citing literature to explain the known dechlorination ability of the selected bacterial genus.
5. The article mentions that the abundance of dechlorinated bacteria in high pollution areas is actually lower, but does not discuss mechanisms such as toxicity threshold or competitive inhibition (such as the inhibition of dechlorinated bacteria by BTEX). Suggest supplementing relevant literature in the discussion or analyzing the relationship between pollutant concentration and dechlorination bacteria abundance through dose-response models.
6. line125 Further improve the soil sampling process, including specific sampling depth, sample weight, preservation, etc
7. line202-203 Improve the meaning of abbreviations “ND”,” S”, and “D”;
8. line 600-602 Verify the reference information.

Author Response

Response to Reviewer 3 Comments

Comments 1: Improve the abstract in the manuscript in the order of Purpose, Methods, Results, and Conclusions

Response 1:Thank you for pointing this out. Revised accordingly.

Comments 2: Suggest reducing the number of keywords

Response 2: Agreed with the point. Two keywords (BTEX degradation; Soil pollution) have been deleted.

Comments 3: Suggest further analysis of the negative correlation between pH and SMC on the dechlorinators, which may be indirectly affected by the redox potential

Response 3: Agreed with the point. Revised accordingly. In general, the redox potential is negatively correlated with pH and SMC.

Comments 4: Suggest supplementing qPCR detection of dechlorination functional genes, or citing literature to explain the known dechlorination ability of the selected bacterial genus

Response 4: Agreed with the point. There references were cited to explain the known dechlorination ability of the selected bacterial genus. （Weatherill et al. 2018; Rekha Seshadri et al. 2005; Ning et al. 2022）

Comments 5: The article mentions that the abundance of dechlorinated bacteria in high pollution areas is actually lower, but does not discuss mechanisms such as toxicity threshold or competitive inhibition (such as the inhibition of dechlorinated bacteria by BTEX). Suggest supplementing relevant literature in the discussion or analyzing the relationship between pollutant concentration and dechlorination bacteria abundance through dose-response models

Response 5: Agreed with this comment. Revised accordingly. Two references were cited to discussion the relationship between pollutant concentration and dechlorination bacteria abundance through dose-response models. (Fan et al. 2022; Zhang et al. 2022).

Comments 6: line125 Further improve the soil sampling process, including specific sampling depth, sample weight, preservation, etc

Response 6: Agreed with this point. Revised accordingly.

Comments 7: line202-203 Improve the meaning of abbreviations “ND”,” S”, and “D”

Response 7: Thank you for pointing this out. We agreed with this comment and have modified the meanings of abbreviations “ND”,” S”, and “D”.

Comments 8: line 600-602 Verify the reference information

Response 8: Agreed with this point. Revised accordingly.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript is well-structured and clearly presents valuable findings on microbial community responses under long-term organic pollution. The use of high-throughput sequencing and correlation analysis provides meaningful ecological insights. The study addresses an important knowledge gap in intrinsic bioremediation potential of legacy-contaminated soils. Overall, it is a nicely written and informative contribution to the field.

Comments:

1. Line 36–38: The statement "It acts not only as a ……….. and pollutant filtration" requires a proper citation to support the multifunctional role of soil in ecosystem services and contaminant filtration.
2. Line 135–136: Did the authors monitor or report the soil temperature at both 0.2 m and 2.2 m depths? Rapid or extreme temperature changes could potentially affect microbial viability and diversity, particularly in deeper layers.
3. Methodology: The section lacks detailed description of how the concentrations of chlorinated aliphatic hydrocarbons (CAHs) such as chloromethane, chloroethene, dichloromethane, chloroform, 1,2-dichloroethane, and BTEX compounds (e.g., toluene, o-xylene, chlorobenzene, 1,2-/1,4-dichlorobenzene) were estimated or quantified. Please elaborate on analytical methods used for pollutant quantification.
4. Line 220: "Figure 1" seems to be a mislabeling and should be corrected to "Table 1" if referring to tabulated data.

Author Response

Response to Reviewer 4 Comments

Comments 1: Line 36–38: The statement "It acts not only as a ……….. and pollutant filtration" requires a proper citation to support the multifunctional role of soil in ecosystem services and contaminant filtration

Response 1:Thank you for pointing this out. We agreed with this comment and cited the relevant references.

Comments 2: Line 135–136: Did the authors monitor or report the soil temperature at both 0.2 m and 2.2 m depths? Rapid or extreme temperature changes could potentially affect microbial viability and diversity, particularly in deeper layers

Response 2: Thank you for pointing this out. We failed to measure the soil temperatures at depths of 0.2m and 2.2m.

Comments 3: Methodology: The section lacks detailed description of how the concentrations of chlorinated aliphatic hydrocarbons (CAHs) such as chloromethane, chloroethene, dichloromethane, chloroform, 1,2-dichloroethane, and BTEX compounds (e.g., toluene, o-xylene, chlorobenzene, 1,2-/1,4-dichlorobenzene) were estimated or quantified. Please elaborate on analytical methods used for pollutant quantification

Response 3: Thank you for pointing this out. Revised accordingly. CAHs, CBs and BETX were determined according to standard protocols outlined by HJ605-2011. The specific pollutant quantification was shown in Table 1.

Comments 4: Line 220: "Figure 1" seems to be a mislabeling and should be corrected to "Table 1" if referring to tabulated data

Response 4: Thank you for pointing this out.We have corrected it to Table 1.

 

Reviewer 5 Report

Comments and Suggestions for Authors

This study investigates the chemical and bacterial properties of soil at a decommissioned pharmaceutical – chemical site in northern Jiangsu Province of China. The researchers selected 3 sites out of 93 soil survey points. The soil samples were collected from two different ranges of depth. The soil microbial communities, physical and chemical properties were estimated using the soil samples collected from deeper parts. The microbial communities were estimated from the surface soils (0.2 m deep). The study compared the presence of dechlorinating bacteria. They found that the relative abundance of dechlorinating bacteria was low and pointed out that the soils have limited intrinsic bioremediation capacity.

The study is intriguing and adds value to determine the bioremediation strategies required to tackle the chemical pollutant in the soil of decommissioned pharmaceutical – chemical site. The investigation is designed and executed nicely. However, it is can be improved to make it more coherent and needs revision. In addition, key citations are required to merit the researchers who developed those methods or identified essential information which are used in this paper. Please refer to the following major and minor comments.

 

Major Comments

1. It is unclear why sites DPS13, DPS14, and DPS16 were selected. Please include either data or cite a particular paper showing the soil contaminants at those sites. (Line 119).
2. From the three sites, surface and deep soil samples were collected. Please include these in the methods with the naming system used. In addition, at certain places data from surface and deep soil samples are combined. For instance D13 represents soils from D13S and D13D in Figure 6E. Please highlight the need for this.
3. Major methods used for chemical analysis and statistical need references. Please include them.

Minor Comments

Line 118 there are more than 18 points in the figure. Is it 93?      

Line 146 Please include a reference for the pH method.

Line 147 and 148 Please include references for the methods used.

Line 164 Please include citation for the reference database (SILVA 138)

Line 169 to 187 Please cite R, and the packages used.

Line 184 Please include references for the bacteria listed as dechlorinating taxa. Is this list exclusive to these organisms or are there other microorganisms (bacteria and fungi) with such properties?

Line 359 “sURFACE”. Please correct this.

 

Overall, the researchers have done a great job of investigating the microbial ecological potential for natural attenuation potential as a bioremediation approach. They identified soil pH and moisture affecting the dechlorinating microbial communities which are comparatively high is surface soil than the deeper soils. They also found cooperative metabolic interactions contributing in the biodegradation of BTEX and chlorinated aliphatic hydrocarbons (CAHs) under natural attenuation conditions. Their findings add value to the understanding of bioremediation strategies. They suggest additional measures to speed up recovery of soil in the study site from the contamination.

 

Author Response

Response to Reviewer 5 Comments

Major Comments

Comments 1: It is unclear why sites DPS13, DPS14, and DPS16 were selected. Please include either data or cite a particular paper showing the soil contaminants at those sites. (Line 119)

Response 1: Thank you for pointing this out. The three sites were selected based on contamination patterns reported in the 2024 soil pollution assessment of the study site. Soil pollutant results of DPS13 and DPS16 exceeded the screening thresholds for Class II 106 land use as defined by the Chinese Soil Environmental Quality Standard GB36600-2018, and the results of DPS14 did not exceed the standard.

Comments 2: From the three sites, surface and deep soil samples were collected. Please include these in the methods with the naming system used. In addition, at certain places data from surface and deep soil samples are combined. For instance D13 represents soils from D13S and D13D in Figure 6E. Please highlight the need for this

Response 2: Thank you for pointing this out. The naming system used was revised accordingly in Table1. Added relevant explanations in Figure 6E: “The point result was the sum of the results of surface soil and deep soil”.

Comments 3: Major methods used for chemical analysis and statistical need references. Please include them

Response 3: Thank you for pointing this out. Soil physicochemical properties were determined according to standard protocols outlined by Lu (1999). CAHs and BTEX were determined according to standard protocols outlined by HJ605-2011.

Minor Comments

Comments 1: Line 118 there are more than 18 points in the figure. Is it 93?

Response 1:Thank you for pointing this out. Revised accordingly.

Comments 2: Line 146 Please include a reference for the pH method

Response 2: Thank you for pointing this out. Relevant reference (Lu, 1999) has been inserted.

Comments 3: Line 147 and 148 Please include references for the methods used

Response 3: Thank you for pointing this out. Relevant reference (Lu, 1999) has been inserted.

Comments 4: Line 164 Please include citation for the reference database (SILVA 138)

Response 4: Thank you for pointing this out. We have attached the citations of the reference database (Quast et al. 2012).

Comments 5: Line 169 to 187 Please cite R, and the packages used

Response 5: Thank you for pointing this out. We have cited R and indicated the R packages used (R Core Team, 2013; Mendiburu, 2019; Oksanen et al. 2008; Ahlmann-Eltze et al. 2021;  Aphalo, 2021; Pohlert et al. 2021; Csardi and Nepusz, 2006; Villanueva and Chen, 2019; Kassambara, 2018).

Comments 6: Line 184 Please include references for the bacteria listed as dechlorinating taxa. Is this list exclusive to these organisms or are there other microorganisms (bacteria and fungi) with such properties?

Response 6: Thank you for pointing this out. Relevant reference (Zhang, 2022) has been inserted. This study did not consider the influence of fungi. Subsequent studies may consider the influence of fungi (including analysis of 18S rDNA).

Comments 7: Line 359 “sURFACE”. Please correct this

Response 7: Thank you for pointing this out. We have corrected the word.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The paper has been well revised and I agree to accept for publication.