Distribution of Microorganisms and Antibiotic Resistance Genes in Production Wastewater During Pumped Storage Power Station Construction

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

This is a very interesting paper dealing with antibiotic resistance in wastewater treatment plants. In my opinion, the article is well done, necessitating, however, some minor modifications, as below I recommend:

1. Line 81 -  indicate the number of retrieved samples and if were collected in the same temporal time, volume, type of recipient.

2. Line 120 - rephrase this paragraph and add more specific data.

 

Comments on the Quality of English Language

Minor English modifications are needeed.

Author Response

Comments 1. Line 81 - indicate the number of retrieved samples and if were collected in the same temporal time, volume, type of recipient.

Response 1:

Thank you for pointing this out. I have provided additional details regarding the sampling process. Mention where in the revised manuscript this change can be found in line 85-88.

 

Comments 2. Line 120 - rephrase this paragraph and add more specific data.

Response 2:

Thank you for pointing this out. I rephrase this paragraph and add more specific data. Mention where in the revised manuscript this change can be found in line 140-144.

Revised:

Line140-144: Calculate the spearman correlation coefficient between environmental factors and species, ARG abundance with “vegan”, “igraph” and “hmisc” packages using R language, and draw the image using origin (2024b). The heatmaps were drawn in origin（2024b）as well, and the lattice value was obtained from -log transformation or the standardized treatment of absolute or relative abundance in the sample.

Reviewer 2 Report

Comments and Suggestions for Authors

The article presents a crucial investigation into the environmental impact of production wastewater generated during the construction of pumped storage power stations (PSPS). By utilizing metagenomic analysis, the study explores the distribution of microorganisms and antibiotic resistance genes (ARGs) in wastewater from two typical PSPS sites. The title, abstract, and introduction part seems fine. However, I suggest to add few recent relevant references in line with your research to validate the proposed idea. I have few doubts about the methodology and results of your research.

1. You mentioned alkaline pH conditions are favored for microbial growth. This is not a new finding. It is already well-established in the literature. 

2. The presence of ARGs linked to common antibiotics (e.g., tetracycline, macrolide, fluoroquinolone) raises significant concerns. The limited effectiveness of wastewater treatment in removing these genes highlights potential environmental and public health risks. This is also established.

3. The Figures are blurred for me. I zoomed in to see the results and compare them with already published studies but couldn't. You must provide high-resolution images. 

I can't find novelty in this article and thus I suggest denying its publication. Or the authors need to highlight the novelty with scientific evidence then I can see in the revised version. 

Comments on the Quality of English Language

minor

Author Response

Comments 1. You mentioned alkaline pH conditions are favored for microbial growth. This is not a new finding. It is already well-established in the literature.

Response 1: 

Thank you very much for raising this question. In this study, a microbial community structure analysis was conducted on water samples under highly alkaline conditions. It was found that when the pH ranged from 10 to 12, the dominant bacterial genus was Serpentinimonas. Additionally, ARGs were also distributed to some extent, providing a foundational basis for further research into potential hosts of resistance genes under these conditions.

 

Comments 2. The presence of ARGs linked to common antibiotics (e.g., tetracycline, macrolide, fluoroquinolone) raises significant concerns. The limited effectiveness of wastewater treatment in removing these genes highlights potential environmental and public health risks. This is also established.

Response 2: 

Previous studies have primarily focused on microbial communities in wastewater from biochemical treatment processes. This study, however, examines wastewater treated only by coagulation and sedimentation. Given that wastewater is reused in municipalities, investigating the microbial community structure and the distribution of antibiotic resistance genes in such wastewater is essential. This research lays the groundwork for future studies.

Revised: 

Line23-25: The results indicate that resistant bacteria and resistance genes can still be present and distributed under the highly alkaline conditions, and the removal efficiency of ARGs by wastewater treatment in PSPS is limited, the attention should be given to the environmental and health risks posed by production wastewater, thereby providing a theoretical basis for the sustainable development of the PSPS industry.

Line202-205: It is worth noting that microbial communities can still exist under highly alkaline conditions, and their abundance significantly increases after treatment. These communities may also become potential hosts for ARGs, posing potential health risks to the environment.

Line242-248: A study have shown that coagulation treatment can remove certain ARGs. However, in this study, the HNEFF sample exhibited 23 more ARG subtypes than the HNINF sample, which is consistent with the findings of Conco ^[32]. Other water samples showed varying degrees of reduction in ARGs, although the reductions were minimal. This suggests that under high alkalinity conditions, the effectiveness of ARG removal may decrease, or alternatively, high alkalinity may promote the horizontal transfer of ARGs.

References:

Na Li, Sheng Guo-Ping, Lu Yong-Ze, et al. Removal of antibiotic resistance genes from wastewater treatment plant effluent by coagulation[J]. Water Research, 2017, 111204-212.

 

Comments 3. The Figures are blurred for me. I zoomed in to see the results and compare them with already published studies but couldn't. You must provide high-resolution images.

Response 3:

Thank you for pointing this out. I will re-upload high-resolution images in the attachment for your review.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript reports bacterial diversity found in the production wastewater. Authors adopted metagenomic approach to detect the bacterial genera and phyla present in the wastewater. Authors also targeted presence of antibiotic resistance genes in the wastewater to understand if this waste can spread resistance markers. Although, authors claimed to use different types of wastewater samples, however, the result section does not provide any details about statistical significance of the presence of resistance markers or bacterial genera. Overall, the manuscript is poorly written with so many language errors. Methods have not been provided with complete details. Some parts of the methods are misleading and it hinted about the lack of sufficient expertise in this field. Specific comments include:

 

·         The authors have not described the need to adopt metagenomic approach in this study. In all, the rationale of this study has not been given clearly.

·         Modify the title. Write antibiotic resistance genes

·         L14-16: Rewrite to avoid meaningless sentences.

·         L16: Write ‘metagenomics’ or ‘metagenome approach’

·         The bacterium, ‘Serpentinimonas’ is an autotrophic bacterium so authors should consider this fact in discussion. Discuss what type of nutrients can be found in this wastewater to feed this bacterium.

·         L23: Write scientifically. ‘..structure of microorganisms’ has other meanings.

·         Introduction does not state clearly how this wastewater can enter to the food chain. Also, the treatment methods for this wastewater should also be introduced here.

·         Section 2.3: This method should be stated with sufficient details. How the DNA was extracted? Which portion/genes of the bacteria were targeted? Was it 16S rDNA based analysis?

·         Regarding the metagenomics of antibiotic resistance markers, describe how these were targeted?

·         Also, in introduction and in discussion, state the rationale behind selection of the ARG markers. In the whole manuscript, authors have not stated even for once the names of the markers which were targeted.

·         L108: Avoid incomplete sentences.

·         L109: this software cannot be used for the alignment of data obtained through metagenomics.

·         L114-116: Provide sufficient details for these analyses.

·         L117-119: Again, BlastP cannot be used for the alignment of gene datasets. This section needs more details.

·         L121-123: Correct its grammar

·         L138-140: State the source of these components and discuss the same

·         L144-145: Avoid incomplete sentence. Besides, revise the following line to bring clarity

·         L176-177: Not all of these pathogens are opportunistic

·         L184: All of these bacteria are free living. The methodology adopted here can only information about the free-living bacteria.

·         Section 3.3: This whole section and other relevant portions need to be rewritten. Antibiotic resistance genes are not ‘free-flowing’ objects. They are part of bacterial genome. Authors cannot consider it as a separate entity without discussing about the source bacterial strains.

·         Discussion is very weak or even non-existent. Authors need to strengthen it.

 

Comments on the Quality of English Language

Extensive language editing is required. There are countless incomplete sentences, verb confusion and redundant expressions.

Author Response

Comments 1: The authors have not described the need to adopt metagenomic approach in this study. In all, the rationale of this study has not been given clearly.

Response 1:

Metagenomic technology is widely used to analyze microbial community structure, function, and their interactions with the environment. It enables the detection and tracking of ARG distribution, which is crucial for environmental health. This study examines wastewater treated by coagulation and sedimentation, with samples exhibiting high alkalinity—an aspect often overlooked in previous research. Investigating the distribution of resistance genes in such water is essential for informing future treatment strategies.

 

Comments 2: Modify the title. Write antibiotic resistance genes

Response 2: 

Thank you very much for your kind reminder. We have made the necessary revisions to the manuscript.

Revised:

Title: Distribution of microorganisms and antibiotic resistance genes in production wastewater during the pumped storage power station construction

 

Comments 3: L14-16: Rewrite to avoid meaningless sentences.

Response 3: 

Thank you for pointing this out. I have rewritten line 14-16 to avoid meaningless sentences.

Line14-16: The construction period of pumped storage power stations (PSPS) generates amounts of production wastewater, which may contain pathogenic bacteria and antibiotic resistance genes (ARGs), potentially posing environmental and health risks.

 

Comments 4: L16: Write ‘metagenomics’ or ‘metagenome approach’

Response 4: 

Thank you for pointing this out. I have already corrected this mistake.

Line16-18: This study uses metagenome approach to analyze the distribution of microorganisms, ARGs and their correlation with water quality indicators in wastewater collected from two typical PSPSs.

 

Comments 5: The bacterium, ‘Serpentinimonas’ is an autotrophic bacterium so authors should consider this fact in discussion. Discuss what type of nutrients can be found in this wastewater to feed this bacterium.

Response 5: 

We have added a note in the manuscript accordingly.

Revised:

Line194-197: And during the construction period, the industrial and domestic wastewater may contain inorganic substances such as carbonates, phosphates, and ammonium salts, which can provide nutrients for autotrophic microorganisms like Serpentinimonas. 

 

Comments 6: L23: Write scientifically. ‘..structure of microorganisms’ has other meanings.

Response 6:

Thank you for pointing this out. We have revised this section of the manuscript accordingly.

Revised:

Line23-28: The results indicate that resistant bacteria and resistance genes can still be present and distributed under the highly alkaline conditions, and the removal efficiency of ARGs by wastewater treatment in PSPS is limited, the attention should be given to the environmental and health risks posed by production wastewater, thereby providing a theoretical basis for the sustainable development of the PSPS industry.

 

Comments 7: Introduction does not state clearly how this wastewater can enter to the food chain. Also, the treatment methods for this wastewater should also be introduced here.

Response 7:

Thank you for pointing this out. I have added the main treatment methods for current production wastewater on line 64-65, and added information about how this wastewater can enter to the food chain on line 75-78.

Revised:

Line64-65: At present, most production wastewater is mainly treated through sedimentation tank or clarifying tank, and the treated wastewater is primarily used for recycling, road dust suppression, washing of stone and irrigation and other purposes.

Line75-78: These ARGs are directly exposed to the environment and can enter the human body through diffusion or bioaccumulation, and further horizontally transfer within the body (such as in the intestine), posing a threat to environmental and health safety^[19].

 

Comments 8: Section 2.3: This method should be stated with sufficient details. How the DNA was extracted? Which portion/genes of the bacteria were targeted? Was it 16S rDNA based analysis?

Response 8:

Thanks for your comments. We have supplemented this section of the manuscript accordingly.

Revised:

Line110-118: After bacterial lysis and DNA extraction using organic solvents, the genomic integrity was assessed by 1% agarose gel electrophoresis. DNA fragmentation (400 bp) was performed using a Covaris M220 ultrasonicator, and a DNA sequencing library was constructed with the NEXTFLEX™ Rapid DNA-Seq Kit. Sequencing was carried out using the Illumina HiSeq 2500 high-throughput sequencing platform with PE150 sequencing. Prior to analysis, raw sequencing data underwent quality control using fastp software. Short reads were assembled with Megahit software, followed by open reading frame (ORF) gene prediction. The predicted gene sequences were clustered using CD-HIT software to construct a non-redundant gene set.

 

Comments 9: Regarding the metagenomics of antibiotic resistance markers, describe how these were targeted?

Response 9: In Section 2.4 of the manuscript, we have supplemented the Methods section.

Revised:

Line133-138: All non-redundant genes were aligned with the Comprehensive Antibiotic Resistance Database (CARD), and the genes of the target species were annotated based on their associated resistance functions. After annotation, the relative abundance of gene sequences corresponding to species and functions was expressed using RPKM (Reads Per Kilobase Million) to eliminate the influence of sequencing depth and gene length.

 

Comments 10: Also, in introduction and in discussion, state the rationale behind selection of the ARG markers. In the whole manuscript, authors have not stated even for once the names of the markers which were targeted.

Response 10: 

In this study, we analyzed only common antibiotic resistance genes and did not focus on any specific resistance gene. Therefore, we aligned the obtained non-redundant genes with the CARD to identify all resistance genes present in the dataset.

 

Comments 11: L108: Avoid incomplete sentences.

Response 11:

Thank you for pointing this out. I have revised the title of Section 2.4 to replace the phrase within the paragraph.

 

Comments 12: L109: this software cannot be used for the alignment of data obtained through metagenomics.

Response 12: 

Thank you for pointing this out. A review of the available data confirms that the BLASTP software can be used to compare and annotate non-redundant genes (proteins) with the CARD. The analysis method was provided by MegaGen Corporation.

 

Comments 13: L114-116: Provide sufficient details for these analyses.

Response 13:

Thank you for pointing this out. However, this analysis method was provided by MegScience, and it can also be found in other studies, such as Comparative metagenome of a stream impacted by the urbanization phenomenon.

 

Comments 14: L117-119: Again, BlastP cannot be used for the alignment of gene datasets. This section needs more details.

Response 14:

Thank you for pointing this out. Similarly, the analysis method was provided by MegScience.

 

Comments 15: L121-123: Correct its grammar

Response 15:

Thank you for pointing this out. I rephrase this paragraph and add more specific data. Mention where in the revised manuscript this change can be found in line140-144.

Revised:

Line140-145: Calculate the spearman correlation coefficient between environmental factors and species, ARG abundance with “vegan”, “igraph” and “hmisc” packages using R language, and draw the image using origin (2024b). The heatmaps were drawn in origin（2024b）as well, and the lattice value was obtained from -log transformation or the standardized treatment of absolute or relative abundance in the sample.

 

Comments 16: L138-140: State the source of these components and discuss the same

Response 16:

Thank you for pointing this out. I added the source of these components and discussion. Mention where in the revised manuscript this change can be found in line 144-145.

Revised:

Line144-145：The above data were processed and analyzed using the MegaGen platform.

 

Comments 17: L144-145: Avoid incomplete sentence. Besides, revise the following line to bring clarity

Response 17:

Thank you for your suggestion. We have made the corresponding revisions in the manuscript.

Revised:

Line168-170: The alpha diversity of species in various wastewater samples was assessed using the Shannon-Wiener Index, Simpson’s Index, and the Chao1 estimator, as presented in Table 3.

 

Comments 18: L176-177: Not all of these pathogens are opportunistic

Response 18:

Thank you for pointing this out. To our knowledge, opportunistic pathogens may exhibit resistance to multiple antibiotics, complicating treatment. Therefore, we use this pathogen as an example to illustrate the distribution of resistant bacteria and resistance genes, which may pose potential health risks.

 

Comments 19: L184: All of these bacteria are free living. The methodology adopted here can only information about the free-living bacteria.

Response 19:

First, we sincerely appreciate your thoughtful feedback. We have made the relevant revisions in the manuscript. Additionally, due to the dual limitations of the water samples and detection methods, we did not perform any analyses on epiphytic bacteria.

Revised:

Line214-219: In addition to potential changes in pH and nutrient composition during the wastewater treatment process, this may also likely due to that some of the pathogens (such as Pseudomonas, Chryseobacterium, and Rhodococcus) were free-living bacteria, it is possible that the structural characteristics of these free-living pathogens make them less likely to be adsorbed and trapped by flocs, resulting in their reduced removal efficiency during the sedimentation process.

 

Comments 20: Section 3.3: This whole section and other relevant portions need to be rewritten. Antibiotic resistance genes are not ‘free-flowing’ objects. They are part of bacterial genome. Authors cannot consider it as a separate entity without discussing about the source bacterial strains.

Response 20:

We have supplemented the discussion in Section 3.3 of the manuscript.

Revised:

Line257-262: A study has shown that Perlucidibaca is a potential host for macrolide resistance genes. In the study by Yongpeng Zhang et al., Limnohabitans was found to be correlated with the abundance of tetracycline and aminoglycoside resistance genes. The co-occurrence patterns of these ARGs suggest that Limnohabitans may play an important role in the environmental dissemination of these resistance genes.

References:

Chunxia Jiang, Diao Xiaoping, Wang Haihua, et al. Diverse and abundant antibiotic resistance genes in mangrove area and their relationship with bacterial communities - A study in Hainan Island, China[J]. Environmental Pollution, 2021, 276116704.

Yongpeng Zhang, Shen Genxiang, Hu Shuangqing, et al. Deciphering of antibiotic resistance genes (ARGs) and potential abiotic indicators for the emergence of ARGs in an interconnected lake-river-reservoir system[J]. Journal of Hazardous Materials, 2021, 410124552.

 

Comments 21: Discussion is very weak or even non-existent. Authors need to strengthen it.

Response 21:

Thanks for your suggestion. We have made revisions to the discussion section in the manuscript.

Revised:

Line238-244: A study has shown that coagulation treatment can remove certain ARGs^[32]. However, in this study, the HNEFF sample exhibited 23 more ARG subtypes than the HNINF sample, which is consistent with the findings of Conco ^[32]. Other water samples showed varying degrees of reduction in ARGs, although the reductions were minimal. This suggests that under high alkalinity conditions, the effectiveness of ARG removal may decrease, or alternatively, high alkalinity may promote the horizontal transfer of ARGs.

References:

Na Li, Sheng Guo-Ping, Lu Yong-Ze, et al. Removal of antibiotic resistance genes from wastewater treatment plant effluent by coagulation[J]. Water Research, 2017, 111204-212.

Line253-258: A study has shown that Perlucidibaca is a potential host for macrolide resistance genes^[35]. In the study by Yongpeng Zhang et al.,^[36] Limnohabitans was found to be correlated with the abundance of tetracycline and aminoglycoside resistance genes. The co-occurrence patterns of these ARGs suggest that Limnohabitans may play an important role in the environmental dissemination of these resistance genes.

References:

Chunxia Jiang, Diao Xiaoping, Wang Haihua, et al. Diverse and abundant antibiotic resistance genes in mangrove area and their relationship with bacterial communities - A study in Hainan Island, China[J]. Environmental Pollution, 2021, 276116704.

Yongpeng Zhang, Shen Genxiang, Hu Shuangqing, et al. Deciphering of antibiotic resistance genes (ARGs) and potential abiotic indicators for the emergence of ARGs in an interconnected lake-river-reservoir system[J]. Journal of Hazardous Materials, 2021, 410124552.

Line223-229: Perlucidibaca has been confirmed as a potential host for ARGs, and Limnohabitans may also serve as a potential host for various resistance genes. Although there are no direct studies linking Malikia to the dissemination of ARGs, considering its membership in the Comamonadaceae family and the role other members of this family play in ARG dissemination, Malikia could also be a potential host for ARGs. Future research may further explore the specific roles and mechanisms of Malikia and other Comamonadaceae members in the spread of antibiotic resistance genes.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

suggest acceptance

Comments on the Quality of English Language

minor

Author Response

Thank you very much.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised version has been improved, however, certain points still need to be considered. 

1. As mentioned in the earlier report, ARG markers are not 'floating objects' in wastewater. DNA (with complete ARGs) cannot remain stable under alkaline conditions (as claimed by the authors for this wastewater). The source of ARGs is bacterial cells. Therefore, in abstract, in introduction and in results, it should be stated correctly. 

2. As authors stressed at least twice in the manuscript that they worked on the subtypes of the ARGs, therefore, a comprehensive list should be provided in the supplementary file. And the same should be discussed with results. 

Comments on the Quality of English Language

There are so many language errors, such as writing methods in present form and redundant expression.

Author Response

1. As mentioned in the earlier report, ARG markers are not 'floating objects' in wastewater. DNA (with complete ARGs) cannot remain stable under alkaline conditions (as claimed by the authors for this wastewater). The source of ARGs is bacterial cells. Therefore, in abstract, in introduction and in results, it should be stated correctly.

Response 1: 

Thank you for raising this point, and we have revised the content of the manuscript accordingly.

Revised:

Line14-16: The construction period of pumped storage power stations (PSPS) generates amounts of production wastewater, which may contain pathogenic bacteria and antibiotic resistance genes (ARGs) in these bacteria, potentially posing environmental and health risks.

Line71-73: This further suggests that reactors in wastewater treatment processes can serve as sources for the spread of antibiotic-resistant bacteria (ARB) and their associated ARGs^[16].

Line234-235: The metagenomic sequence data were compared with the CARD database for a comprehensively screening of ARGs carried by microorganisms in wastewater.

Line344-346: This study used metagenomic sequencing technology to analyze the microbial community structure and the distribution characteristics of ARGs carried by them both in correlation with water quality indicators in wastewater of PSPSs.

 

2. As authors stressed at least twice in the manuscript that they worked on the subtypes of the ARGs, therefore, a comprehensive list should be provided in the supplementary file. And the same should be discussed with results.

Response 2:

Thank you very much for your suggestion. We have addressed the ARG subtypes in Section 3.3 of the manuscript.

Revised：

Line250-254: It is noteworthy that 192 ARG subtypes were detected under high alkalinity conditions, while Li et al.^[34] conducted a statistical analysis of ARG distribution across ten different environments and identified 260 ARG subtypes. This indicates that a single environment still exhibits a high distribution of ARG subtypes under the conditions of this study(Supporting Information 1).

Line256-262: Taking the tet gene as an example, the abundance of this gene in effluent under high alkalinity conditions is higher than that in influent. Furthermore, in the SS environment, the abundances of this gene subtype in both influent and effluent show a certain degree of reduction. This indicates a correlation between the tet gene and water quality, a finding that is consistent with the research of Lin et al.^[35] Therefore, it can be inferred that different effluent water qualities exert varying degrees of influence on the distribution of ARG subtypes. Future studies could further investigate the underlying mechanisms affecting this relationship.

Reference：

Bing Li, Yang Ying, Ma Liping, et al. Metagenomic and network analysis reveal wide distribution and co-occurrence of environmental antibiotic resistance genes[J]. The ISME Journal, 2015, 9(11): 2490-2502.

Xiaojun Lin, Ruan Jingjing, Huang Lu, et al. Comparison of the elimination effectiveness of tetracycline and AmpC β-lactamase resistance genes in a municipal wastewater treatment plant using four parallel processes[J]. Ecotoxicology, 2021, 30(8): 1586-1597.

 

Comments on the Quality of English Language

There are so many language errors, such as writing methods in present form and redundant expression.