Impact of Effective Microorganisms and Chlorella vulgaris on Eriocheir sinensis and Water Microbiota in Ponds Experiencing Cyanobacterial Blooms

Round 1

Reviewer 1 Report

Journal: Sustainability (ISSN 2071-1050)

Manuscript ID: sustainability-2330531

Type: Article

Title: Effects of Effective Microorgnisms and Chlorella vulgaris on Eriocheir sinensis and Water Microbiota in Ponds Experiencing Cyanobacterial Blooms

Section: Sustainable Water Management

Special Issue: New Trends and Perspectives in Sustainable Aquaculture

 

1. Introduction

Lines 39-41

“In E. sinensis culture ponds, cyanobacterial blooms are commonly formed by the following genera, e.g. Microcystis, Cyanobium, Calothrix, Sphaerospermopsis and Cylindrospermopsis [1].”

Latin scientific names, including Microcystis, should be italic.

2. Lines 41-44

“It is well documented that cyanobacterial blooms and their secondary metabolites microcystins can cause a series of problems in aquaculture, such as compromising water quality, toxic to aquatic animals, and even threatening human health by bioaccumulation through food chains [5-8].”

Microcystins are not the only, mainly or necessarily the major toxic components in cyanobacteria. Cyanobacteria can synthesize and release a wide range of other toxic metabolites including anatoxin-a, retinoic acids. Please read and cite the following paper.

Challenges of using blooms of Microcystis spp. in animal feeds: A comprehensive review of nutritional, toxicological and microbial health evaluation. https://doi.org/10.1016/j.scitotenv.2020.142319

3. Materials and Methods

Lines 67-68

“This experiment was conducted from July 19th to July 26th, 2021 in Yangzhong scientific research base of Freshwater Fisheries Research Center, CAFS.”

Please insert the full name of CAFS, Chinese Academy of Fishery Sciences.

4. Line 90

“Microbial samples were collected as described in the previous study [1].”

Please insert some details in the revised manuscript.

5. Lines 112-114

“The V3-V4 region of the 16S rDNA and the V4 region of the 18S rDNA were amplified by PCR as described in the previous study [1].”

Please list the sequences of primers used in the revised manuscript. The primers can affect the PCR amplification and information of species.

6. Fig. S2C, Table 3C

Lines 230-233

“In addition, the NG7 group had an increased richness and evenness of phytoplankton compared to the NG0 group, while the CS7/CN7 group had a decreased richness and evenness of phytoplankton compared to the CS0/CN0 group (Fig. S2C).”

Lines 241-243

“Interestingly, the Simpson diversity of phytoplankton was decreased in the NG7 and CS7 groups than their initial levels, while it had no significant difference between the CN0 and CN7 groups (Table 3C).”

Which phytoplankton did you analyze? Prokaryotic, eukaryotic or both? Please insert more details in the revised manuscript.

7. Please insert the data of cyanobacteria in three groups, including the control group (NG), the supplement group of effective microorgnisms (EM) and Chlorella vulgaris (CS) and the non-supplement group (CN). The data of cyanobacterial density, biomass, community composition and changes during the experiment is very important for this study.

8. Please add some analysis of relationship between cyanobacteria and water quality, microbiota, growth, digestive and antioxidant ability of crabs.

9. Any toxic cyanobacteria or cyanotoxins in the ponds? As mentioned above, cyanobacteria can synthesize and release a wide range of toxic metabolites, which can affect the aquatic system, including phytoplankton, bacteria, fungi and crabs. Please add some discussion.

Author Response

Thank you very much for your support and affirmation of this paper. According to your constructive and detailed advices, we have carefully revised our manuscript, and the revision was shown below. We believe your advices will improve our paper.

Author Response File: Author Response.docx

Reviewer 2 Report

The freshwater Chinese mitten crab (E. sinensis) is an important crustacean species that is widely cultivated in most provinces of China and has a high economic and nutritional value. The authors suggest a method to to prevent and control of potential risks of cyanabacteria in the culture of E. sinensis.

In material methods they usually cited their previous paper. It may be good idea to give the name of the method without giving details. For example X method was done, for details please see our previous paper.

7 days experiment time is the weak point of the study.  Authors can  add an explanation how they decided experiment time and what is the growth stage of crabs in experiment. 

The authors can also add a paragraph into the introduction or conclusion, that emphasise the important of the study like below:

"However, the presence of cyanotoxins and water pollution could cause undesirable chemicals  to become residual, especially in the hepatopancreas (the main edible part), thereby reducing the quality of E. sinensis Therefore, developing more sustainable models of E. sinensis breeding is critical".

The manuscript focused on E. sinensis and seems local. Please add a paragraph into the discussion part that explains how your findings can be conducted in aquaculture or other similar aquatic organisms. 

The manuscript can be accepted for publication after minor revision. Please see the attachment for other comments. 

 

Comments for author File: Comments.pdf

Author Response

Thank you very much for your support and affirmation of this paper. According to your constructive and detailed advices, we have carefully revised our manuscript, and the revision was shown below. We believe your advices will improve our paper.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors studied the interactions and community assembly in different ponds that are experiencing cyanobacterial blooms. They found that effective microorganisms combined with C. vulgaris promoted the restoration of the bacterial and fungal community composition in cyanobacterial blooms ponds, and thus provided insight for the prevention and control of potential risks in aquaculture. In general, the experimental design of this paper is reasonable, the results were well presented. However, the introduction and the method sections need significant improvement. This paper should also reference more papers about cyanobacterial and associated EM interactions, instead of all focus on algae. I suggest reconsider after major revision.

 

Please see my detailed comments below:

 

1.     Line 43: please add cyanobacterial blooms also affect the microbial community assembly.

 

Wang, K., Razzano, M., & Mou, X. (2020). Cyanobacterial blooms alter the relative importance of neutral and selective processes in assembling freshwater bacterioplankton community. Science of the Total Environment, 706, 135724.

 

2.     Line 45: you were talking about cyanobacterial bloom but suddenly jump into algae. I would suggest adding several papers about the cyanobacterial and associated microorganisms interactions using the following papers. then transit to algae to make your intro more comprehensive. Same for discussion, also reference more papers about interactions between cyanobacteria and associated microorganisms.

 

Wang, K., Mou, X., Cao, H., Struewing, I., Allen, J., & Lu, J. (2021). Co-occurring microorganisms regulate the succession of cyanobacterial harmful algal blooms. Environmental Pollution, 288, 117682.

 

Woodhouse, J. N., Ziegler, J., Grossart, H. P., & Neilan, B. A. (2018). Cyanobacterial community composition and bacteria–bacteria interactions promote the stable occurrence of particle-associated bacteria. Frontiers in Microbiology, 9, 777.

 

Wang, K., & Mou, X. (2021). Coordinated diel gene expression of cyanobacteria and their microbiome. Microorganisms, 9(8), 1670.

 

 

 

3.     Line 90: to confirm, the in situ parameters were not measured the same time as sampling? if so, any concern about the difference between different time?

 

4.     Line 112: can you also add the primers sequences here. You can reference your previous published paper but not make sure you have enough info here. otherwise, your readers will have to go to your previous paper back and forth when reading this paper. sane for other places.

 

5.     Line 125: more details here, what's the filtering criteria, such as score, length, etc.

 

6.     Line 126: list the specific filtering conditions.

 

7.     Line 130: what's your criteria to select the repreetative sequence?

 

8.     Line 139: the taxa abundance matri may be a very sparse matrix. did you do any process before data analysis?

 

9.     Line 140: you meant the absolute value of 4?

 

10.  Line 152: any transformation for those parameters that do not satisfy parameteric tests?

 

11.  Line 159: spell it out when first time using it.

 

12.  Line 163: add a space between the number and the unit. same for other places.

 

13.  Line 180: what's unit for different parameters? please add that in this table

 

14.  Line 209: I would suggest add another short paragraph here to show your sequence depth, and how many seqs passed quality control, etc.

 

15.  Line 252: You did not mention PCoA analysis in the statistical analysis section. please add to that section. same for other statistical analysis used in this paper.

 

Author Response

Thank you very much for your support and affirmation of this paper. According to your constructive and detailed advices, we have carefully revised our manuscript, and the revision was shown below. We believe your advices will improve our paper.

Author Response File: Author Response.docx

Reviewer 4 Report

The problem of cyanobacterial blooms in aquaculture is growing as a result of climate change. Therefore, the subject matter raised by the authors of the manuscript fits well into the current research needs and challenges of the modern world.   The authors supported their theses with advanced tools in the field of molecular biology. As a result, they characterized in detail the microbial variability. I highly appreciate the research methods used. However, the short time of observations may be a weak point.
  The authors assessed the potential combined effect of EM and C. vulgaris on the prevention and control of cyanobacteria in an E. sinensis breeding pond. As a result, the addition of EM and C. vulgaris improved water quality in the E. sinensis breeding pond by reducing nitrogen levels. Supplementation with EM and C. vulgaris was found to improve the effect on bacterial diversity and equality, while inhibition of the effect on diversity in fungal and phytoplankton communities was associated with supplementation with EM and C. vulgaris. sinensis joint. The following results, although they are limited in the form of local character, can be an inspiration for research for scientists dealing with other species.   In view of the above, I positively evaluate the entire manuscript and recommend its acceptance for further stages of the publishing procedure.        

 

Author Response

Thank you very much for your support and affirmation of this paper. We are encouraged by your comments. We will continue to carry out relevant research in the future.

We agree that the short time of observations may be a weak point. The experiment time was selected based on the outward appearance of pond water. In the CS group, the cyanobacterial blooms were significantly reduced, with an obvious decreasing of emerald green film at the downwind of the ponds on the seventh day of the experiment. Therefore, we set 7 days as the experimental period in the present study. Next, we will conduct a long-term study in explanation the combined effect of EM and C. vulgaris.

Round 2

Reviewer 1 Report

Journal: Sustainability (ISSN 2071-1050)

Manuscript ID: sustainability-2330531-peer-review-v2

Type: Article

Title: Effects of Effective Microorgnisms and Chlorella vulgaris on Eriocheir sinensis and Water Microbiota in Ponds Experiencing Cyanobacterial Blooms

Section: Sustainable Water Management

Special Issue: New Trends and Perspectives in Sustainable Aquaculture

 

The manuscript improved during the revisions. However, there are still some issues need to be addressed. I have the following comments and suggestions for the authors to improve the quality of manuscript.

 

1. Section “2.8 Statistical analysis”

Lines 181-183

“Since 181

log10-transformed data did not coincide with normal distribution, robust one-way ANOVA was used for non-normally distributed data.”

This is wrong. if your data is very non-normal, it would be worth using a non-parametric test. Please re-analyze you data.

2. “3.5 Community composition”

Lines 311-312

“In the present study, the dominated cyanobacterial genus was Microcystis PCC-7914, accounting 83.54% and 99.12% in the CS0 group and CN0 group, respectively (Fig. S3).”

How did you get data of cyanobacteria? Please add the details in the revised manuscript.

3. Fig. S3

How did you calculate relative abundance of dominant cyanobacterial genus in each group. Please present the details and the formula in the figure captions.

Author Response

Thank you very much for your suggestions and comments, which improved our manuscript significantly. We believe that these changes have made this manuscript a quality paper.  Please see the attached file for details.

Author Response File: Author Response.docx

Reviewer 3 Report

I'm good with the revision. just one minor comment before publication. reference 12 and 13 are the same. Suggest the author double-check.  

Author Response

Thank you very much for your support and affirmation of this paper. As suggested, we carefully checked our manuscript and deleted the duplicate references.