The Microbial Community Composition and Nitrogen Cycling Metabolic Potential of an Underground Reservoir in Rizhao, Shandong Province, China

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript and giving us a chance to revise our manuscript and resubmit it to Water. We are very grateful to the comments and helps you provided. We have deleted irrelevant references and reorganized references. We have thoroughly revised our manuscript according to all the provided comments and suggestions. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Our responses to your comments and suggestions are provided below.

Are all the cited references relevant to the research?

Must be improved

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In my opinion, the biggest problem with the manuscript is that the logic is unclear, which is reflected in many aspects. The first is the abstract, which does not highlight the main findings and conclusions. For example, "Phyla Proteobacteria dominated the bacterial community in raw water, while and archaea Thaumarchaeota and bacteria Proteobacteria dominated the bacterial community in undergournd reservoir", these phyla are recognized as the most common and the most abundant in freshwater river, lakes or reservoirs, so it is not necessary to write in the abstract.

The problem of unclear logic also exists in the introduction. For example, lines 57-59, I cannot understand how this sentence relates to this study. In lines 86-88, “Given the relative complexity of nitrogen conversion, studying the nitrogen cycling state of underground reservoirs is crucially necessary”. This is a false statement. We study the nitrogen transformation because of its importance, not because of its complexity.

Response 1: Thank you very much for your comments and suggestions. We have refreshed the logic of the entire paper and revised the issues you pointed out. We have refined the depth of analysis of the phylum Proteobacteria down to the subphylum, and then did find a lot more interesting conclusions. For the relative abundance of bacterial communities, Gammaproteobacteria dominated the bacterial community in raw water, while Gammaproteobacteria and Alphaproteobacteria dominated the bacterial community in underground reservoir water. In addition, the relative abundance of Nitrospirae was observed to be noticeably higher in the underground reservoir water. We rewrote the abstract and the corresponding results and discussion sections, all of the revised parts have been labelled in the manuscript. Also, the lines 57-59 was removed, and the lines 86-88 has been revised.

“Given the crucial position and intricate nature of nitrogen conversion, it is imperative to explore the nitrogen cycling state of underground reservoirs, particularly considering that the nitrogen cycling metabolic potential of such reservoirs remains unknown”

Comments 2: Section 2.1. The study compared the microbial communities and physicochemical differences in the Rizhao Reservoir and an underground reservoir. The author should clearly explain the relationship between these two reservoirs. Are they connected? Which reservoir is upstream?

Response 2: We appreciate these comments and suggestions. We have added the relevant information in section 2.1.

“The study area is situated in Rizhao City, Shandong Province, China (35°25'34"N-35°26'51"N, 119°20'51"E-119°21'51"E), with the main river being the Futong River. The Futong River is a seasonal river that flows in a southeasterly direction and eventually empties into the Yellow Sea (Figure 1). The Rizhao Reservoir is positioned upstream of the Futong River, and approximately 5 km downstream of the Rizhao Reservoir lies the Futong underground reservoir. Constructed in June 2022, the Futong underground reservoir boasts a total capacity of 9,606,000 m³. The primary water source for the Futong underground reservoir is derived from the outflow of the Rizhao Reservoir, supplemented by precipitation recharge and groundwater recharge.”

Comments 3: Section 3.1. High-throughput sequencing methods cannot perform absolute quantification of sequences, so the differences in sequences between different samples are meaningless. And the sequence has been sub-sampled, so the number of sequences should be clipped by the minimum number.

Response 3: We are sorry for this ambiguity. We did make subsamples, which we have clarified in the revised manuscript.

“At a 97% similarity level, the total Operational Taxonomic Unit (OTU) richness was 1,297 for bacterial communities and 573 for microeukaryotic communities across all samples after clipped by the minimum sequence number of each group.”

Comments 4: L.196-199, how was this result obtained? This study did not compare changes in physicochemical parameters of underground reservoirs at different times.

Response 4: We are sorry for this ambiguity again. We are referring to the fact that previous research has found something about this, not our research. We have clarified this question in the revised manuscript.

“Previous studies have indicated that the water quality of underground reservoirs tends to degrade over time, potentially attributed to increased deep-layer oxygen depletion and the influx of limiting nutrients for algae growth, whcih could lead to a heightened risk of algal blooms and eutrophication.”

Comments 5: L204-205, Shannon index, Simpson index and OTU richness are not the main indicators of evenness.

Response 5: Thank you very much for your comment. We have revised the statement according to your suggestions.

“Alpha diversity indices were employed to thoroughly evaluate species richness, and the values for alpha diversity were determined for each individual sample. Subsequently, the average and standard deviation of alpha diversity were calculated (Table 2). Notably, both microbial community richness (OTU richness) and microbial community diversity (Simpson index) were found to be significantly lower in the underground reservoir, en-compassing both bacterial and microeukaryotic communities (one-way ANOVA, P > 0.05) (Table 1). Despite these differences, the Shannon index exhibited comparable values be-tween the raw water microbial communities and underground reservoir microbial com-munities.”

Comments 6: Mantel test can only reflect the correlation between microbial communities and physical and chemical parameters, but not reflect the causal relationship between them. The expression of L224-233 is not rigorous, please rewrite it.

Response 6: Thank you very much for your comment. We have revised the statement according to your suggestions.

“To assess the potential impact of environmental factors on microbial communities, a Mantel test was employed to examine the correlation between bacterial and microeukaryotic communities and the detected environmental factors (Table. 3). The results indicated that both bacterial and microeukaryotic communities were significantly correlated with environmental factors (P < 0.001). Moreover, the bacterial community demonstrated great-er sensitivity to environmental factors compared to the microeukaryotic community (bac-terial community r=0.786, microeukaryotic community r=0.724). For bacterial communities, the correlation of DO (R=0.437, P = 0.018), TN (R=0.747, P < 0.001), TP (R=0.707, P < 0.001), NH4+-N (R=0.594, P = 0.009) and Chl a (R=0.672, P < 0.001) were significant, while TN (R=0.623, P < 0.001), TP (R=0.597, P < 0.001), NH4+-N (R=0.375, P = 0.041) and Chl a (R=0.528, P = 0.005) correlated with the microeukaryotic communities more significantly. The pH, NO3--N and NO2--N showed no significant correlation with both bacterial and microeukaryotic communities.”

Comments 7: L243-246, this statement is too narrow. Please rewrite it.

Response 7: Thank you very much for your suggestions. We have revised the statement according to your suggestions.

“The impact of water nutrient levels on microbial communities is particularly noteworthy, as elevated nutrient levels can directly influence the composition and distribution of water microbial communities. Furthermore, nutrient levels can affect the composition and abundance of heterotrophic flagellates and ciliates, as well as the biomass and distribution characteristics of algae, consequently influencing the overall diversity of microbial communities.”

Comments 8: Archaea and Bacteria should be described separately. Archaea should not be considered as part of Bacteria. L261-263, Microeukaryotes are not Bacteria.

Response 8: Thank you very much for your comments. We analysis the bacteria and archaea separately in the revised manuscript.

Comments 9: Figure 5, the meaning of the “*” should be added.

Response 9: Thank you very much for your suggestions. We have added the relevant information.

“Significant differences are indicated by * (one-way ANOVA, *** P < 0.001; ** P < 0.01; * P < 0.05). ”

Comments 10: The abstract and conclusion should not be repeated.

Response 10: Thank you very much for your comments. We have rewrite the abstract and conclusion part to avoid their repeating.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1: We have had our manuscript checked by a colleague fluent in English writing.

5. Additional clarifications

None.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript and giving us a chance to revise our manuscript and resubmit it to Water. We are very grateful to the comments and helps you provided. We have thoroughly revised our manuscript according to all the provided comments and suggestions. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Our responses to your comments and suggestions are listed below.

Are all the cited references relevant to the research?

Can be improved

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

The article presents a very nice description of possible nitrogen pathways in the underground water reservoir.

However, the nice results are opaqued by a very poor introduction to the problem of such reservoirs, which are not, according to my knowledge of a graduate water technology engineer, well known outside very specific areas (e.g., China) and even less to microbial ecologists (and I think, I have been invited as such reviewer). I lack more information about such waterworks and a discussion of their "environmental" conditions (e.g., lack of light and its role is not discussed as well while Chl_a concentration is rising there). I've found very relevant information in (the MDPI article) https://doi.org/10.3390/ijerph16111921, while the manuscript cited articles are not free-available.

Response 1: Thank you very much for your comments and suggestions. We have added the relevant information in section 2.1.

“The study area is situated in Rizhao City, Shandong Province, China (35°25'34"N-35°26'51"N, 119°20'51"E-119°21'51"E), with the main river being the Futong River. The Futong River is a seasonal river that flows in a southeasterly direction and eventually empties into the Yellow Sea (Figure 1). The Rizhao Reservoir is positioned upstream of the Futong River, and approximately 5 km downstream of the Rizhao Reservoir lies the Futong underground reservoir. Constructed in June 2022, the Futong underground reservoir boasts a total capacity of 9,606,000 m³. The primary water source for the Futong underground reservoir is derived from the outflow of the Rizhao Reservoir, supplemented by precipitation recharge and groundwater recharge.”

Comments 2: As I mentioned above, the nitrogen pathways are described in a nice way, which contrasts with a very basic level of information on present prokaryotes and eukaryotes. Why should be mentioned the "famous species" of Proteobacteria if only phylum level was investigated? The information on eukaryotes is even poorer and cited literature on them is very limited to more technical and regional literature. E.g., dinoflagellates are mentioned as important phytoplankton (correctly) dominated in the raw water with minimum Chl a concentration but not important in the reservoir. However, the well-defined taxon includes both phototrophs, mixotrophs, heterotrophs, and parasites that are not distinguished in your results, i.e., of very low indication level. The definition of ochrophytes is even more general and their higher importance in seawater is supported by the cited article dealt with a hydrological model of an aquifer, very useful for the present work, but I do not agree that it is an authority to state such conclusion from the microbiological point of view.

Response 2: Thank you very much for your comments and suggestions. We have refined the depth of analysis of the phylum Proteobacteria down to the subphylum, and then did find a lot more interesting conclusions. For the relative abundance of bacterial communities, Gammaproteobacteria dominated the bacterial community in raw water, while Gammaproteobacteria and Alphaproteobacteria dominated the bacterial community in underground reservoir water. In addition, the relative abundance of Nitrospirae was observed to be noticeably higher in the underground reservoir water. For eukaryotic microorganisms, we intend to study them at the level of phylum in this article, while they will be targeted in subsequent studies (experiments are in progress).

“The Figure 4 displays the relative abundances of bacterial and microeukaryotic communities at the phylum level. For the relative abundance of bacterial communities, Gammaproteobacteria dominated the bacterial community in raw water, while Gammaproteobacteria and Alphaproteobacteria dominated the bacterial community in underground reservoir water. Gammaproteobacteria is widespread in the water environment, including many pathogenic bacteria, such as Escherichia coli, Salmonella, Vibrio cholerae, Helicobacter pylori and other famous species. A previous study showed that the relative abundance of Gammaproteobacteria was high in both mesotrophic and eutrophic water environment, which was also observed in our investigation. In addition, the relative abundance of Nitrospirae was observed to be noticeably higher in the underground reservoir water, as Nitrospirae was identified as one of the most predominant nitrite-oxidizing bacteria in the ocean. For average relative abundance of microeukaryotic communities, Dinoflagellata, Rotifera and Ochrophyta dominated the microeukaryotic community in raw water, while Ochrophyta and Choanoflagellida dominated the microeukaryotic community in underground reservoir. Dinoflagellate is an important basic part of the food chain in modern oceans, and it is also a major oxygen producer. It also widely exists in freshwater and brackish lakes and other water bodies. Ochrophyta mostly live in sea water bodies along the coast of the mainland, and are rare in fresh water. Ochrophyta were dominant species in raw water and underground reservoir, indicating that there may be seawater intrusion in this area.”

Comments 3: Nitrogen pathways look better documented but also in their case, I guess there are publications published before and/or using a more biochemical point of view. Browsing within the cited literature, a wetland is hardly comparable to your system. Who is fixing nitrogen in the underground reservoir?

Response 3: Thank you very much for your comments and suggestions. We find more relevant literature to support our findings. We have remove the relevant contents about “who is fixing nitrogen in the underground reservoir” as we can't be sure of that conclusion yet. We will delve deeper into this question in the follow-up study.

“Our findings reveal a significant reduction in the metabolic potential of microbial nitrogen functional genes in various pathways of the nitrogen cycle, with the exception of the amoA functional gene. The nitrogen cycle in water systems is primarily orchestrated by microorganisms through redox processes, encompassing mineralization, nitrogen fixation, denitrification, nitrate reduction, nitrate dissimilatory reduction to ammonium, nitrogen assimilation, ammonia oxidation, nitrification, and anaerobic ammonia oxidation. Two key processes within the nitrogen cycle are nitrification and nitrate reduction. The initial stage of nitrification involves the conversion of NH₄⁺-N to NO₂⁻-N, a crucial rate-limiting step. Autotrophic ammonia-oxidizing bacteria and ammonia-oxidizing archaea are the primary functional microorganisms involved in this process, with the ammonia monooxygenase gene serving as a key molecular marker. The ammonia monooxygenase gene involved in catalysis consists of three subunits, amoA, amoB, and amoC. Quantitative studies of ammonia oxidizing microorganisms typically use amoA as a molecular marker. Notably, amoA, one of the subunits of the ammonia monooxygenase gene, showed a significant increase in metabolic potential, indicating heightened nitrification activity in the underground reservoir. Hou et al. found that the diversity of ammonia oxidizing bacteria in the Taihu Lake and Chaohu Lake, two large eutrophic lakes in China, increased from mesotrophic to eutrophic, and was positively correlated with the number ratio of ammonia-oxidizing bacteria and ammonia oxidizing archaea. Our results are consistent with higher concentration of TN, TP, NO₃^--N, NO₂^--N, but lower concentration of NH₄⁺-N in underground reservoir. Furthermore, the abundance of Thaumarchaeota in the underground reservoir is also consistent with these findings. As Thaumarchaeota is known for their role in ammonia oxidation, likely contribute to the increased nitrification potential observed in the underground reservoir. ”

4. Response to Comments on the Quality of English Language

Point 1:

Response 1: We have had our manuscript checked by a colleague fluent in English writing.

5. Additional clarifications

None.