Gas Chromatography-Mass Spectrometric Study of Low-Molecular-Weight Exogenous Metabolites of Algae-Bacterial Communities in the Laboratory Accumulative Culture

Round 1

Reviewer 1 Report

This manuscript presents a scientific study that employs gas chromatography-mass spectrometry to investigate the exogenous metabolites in the accumulated culture of algae-bacterial communities from natural river water. The research focuses on the identification and characterization of specific metabolites within these communities, shedding light on their metabolic processes and potential ecological significance. The following comments should be addressed prior to further consideration.  

Introduction Section:

1.To engage the readers' interest, improvements were required in the introduction, such as citing significant research findings or posing thought-provoking questions to introduce the article's topic.

2.The introduction should more clearly articulate the specific aims and questions of the research. Readers should have a clear understanding of what the core issue of this study is and why it is significant for ecology and aquatic biology.

3.Additionally, elucidate the innovative aspects of this study compared to other related research more clearly to highlight its uniqueness.

Materials and Methods Section:

1.On page 3, lines 114-115, please explain how the ratio of river water to the nutrient medium was determined and why this ratio was chosen.

2.Regarding the three testing stages, provide a clearer explanation of how these stages were determined and why a two-week interval was chosen.

3.On page 4, lines 149-152, elaborate on the choice of using morphological characteristics for the identification of algae and bacteria and discuss the accuracy and reliability of this method.

4.On page 3, lines 121-123, discuss why parallel experiments were not conducted, as this could enhance the credibility and accuracy of the experimental results.

Results Section:

1.When describing the experimental results, consider starting with an overall summary, listing the various categories of substances and their general variations. Then, proceed to analyze each substance individually to make the results more lucid.

2.In regard to the contentious substances mentioned on page 9, lines 209-210, contemplate conducting further experimental research to ascertain whether their synthesis and decomposition are due to light exposure and time or are the result of microbial and algal metabolic functions.

3.It is recommended to add some figures and tables in the Results section, such as records of the experimental process or micrographs of the microbes and algae. This will enrich the experimental content and enhance the credibility of the results.

4.Adding subheadings within the Results section for better segmentation would improve the overall structure of the article.

Conclusion Section:

1. Emphasize the significance of ecological and biochemical mechanisms: In the conclusion, emphasize the significance of these findings in the context of ecological and biochemical mechanisms in freshwater ecosystems.

2.Clearly state future research directions: Explicitly mention the potential areas of further research in this field within the conclusion.

3.Discuss the potential practical implications: The conclusion should encompass the potential practical implications of these findings. How can this research provide valuable information for environmental management, water quality control, or other practical applications?

In addition, the manuscript's References format requires careful revision.

Moderate language level 

Author Response

Comments and Suggestions for Authors

This manuscript presents a scientific study that employs gas chromatography-mass spectrometry to investigate the exogenous metabolites in the accumulated culture of algae-bacterial communities from natural river water. The research focuses on the identification and characterization of specific metabolites within these communities, shedding light on their metabolic processes and potential ecological significance. The following comments should be addressed prior to further consideration.  

We would like to thank the reviewer for comments.

Introduction Section:

1. To engage the readers' interest, improvements were required in the introduction, such as citing significant research findings or posing thought-provoking questions to introduce the article's topic.

The introduction is logically structured and revised.

2. The introduction should more clearly articulate the specific aims and questions of the research. Readers should have a clear understanding of what the core issue of this study is and why it is significant for ecology and aquatic biology.

We have expanded the scope of the work.

The objective of the research is to carry out a gas-chromatography-mass spectrometric study to qualify and quantify the composition of dissolved LMWOCs produced by cyanobacteria and microalgae using an experimental microecosystem cultivated for 1.5 months at three time points with a fortnight interval.

3. Additionally, elucidate the innovative aspects of this study compared to other related research more clearly to highlight its uniqueness.

We agree. The paper is added.

The innovation of the research is the study of ecological features of the culture of cyanobacteria and microalgae in dynamics by modern gas-chromatography-mass spectrometric methods.

The uniqueness of the experiment is associated with the fact that it cannot be repeated because of water taken from a natural ecosystem, a river, with chemical and algal compositions constantly changing. The chemical composition of exogenous metabolites of phototrophs was studied in the laboratory since hexane-based extraction does not destroy cells. We have indirect evidence of interactions between the components of algal-bacterial communities, namely, LMWOCs qualitative and quantitative composition changes along with simultaneous changes in the composition and number of microorganisms – members of the algae-bacterial system.

Materials and Methods Section:

1. On page 3, lines 114-115, please explain how the ratio of river water to the nutrient medium was determined and why this ratio was chosen.

To obtain an enrichment culture, BGN-11 medium was added to the water from the analyzable reservoir.  According to numerous reported data medium BGN-11 is one of the most promising for cultivating cyanobacteria and building-up large biomass. The culture was placed in a 1L cylinder, where 750 ml of the reservoir water and 250 ml of medium BG-11 (1/4) were introduced. There are different ratios reported to produce enrichment cultures: 1/4, 1/5, 1/10, 1/100. We focused on the ratio of ¼ as one allowing intensive development of cyanobacteria and algae in the presence of the nutrient medium.

2. Regardingthethreetestingstages, provide a cleare rexplanationofhowthesestagesweredeterminedandwhya two-week interval was chosen.

.Cyanobacteria and microalgae enter the station ary growthp hase from the 6^th day to 20^th depending on culture conditions. Sowe considered anaverage time interval of 14 days during which differences in the metabolite composition are getting noticeable.

3. On page 4, lines 149-152, elaborate on the choice of using morphological characteristics for the identification of algae and bacteria and discuss the accuracy and reliability of this method.

According to Kostikov et al. (2001) the accuracy of the determination of algae in mixed soil cultures at the generic level is 80-90%, whereas at the species level is only 20-40%. For identification cultures were purified and brought to algologically pure strains. Taxonomic diagnostics was carried out on the basis of cultural, physiological, biochemical, and morphological properties of algae and cyanobacteria. These methods are conventional and are based on identification by phenotypic traits. But at present in order to compare algae and cyanobacteria molecular procedures are needed, taking into account cell ultrastructure and morphology. In our further research, we are going to taxonomize them in a complex.

4. On page 3, lines 121-123, discuss why parallel experiments were not conducted, as this could enhance the credibility and accuracy of the experimental results.

Discussed. We actually explored a unique ecosystem that had its own unique succession. Even if a parallel experiment had been set up, it would have been a different ecosystem, which, as a result of the influence of stochastic factors, could have received a different direction of development, possibly very different from another ecosystem. In this case, we would be dealing with a different algobacterial community and possibly a different set of metabolites.

Results Section:

1. When describing the experimental results, consider starting with an overall summary, listing the various categories of substances and their general variations. Then, proceed to analyze each substance individually to make the results more lucid.

We agree. Redone in the text and highlighted in color.

2. In regard to the contentious substances mentioned on page 9, lines 209-210, contemplate conducting further experimental research to ascertain whether their synthesis and decomposition are due to light exposure and time or are the result of microbial and algal metabolic functions.

There is a discussion in the text.

As to phthalates, it is necessary to consider the possibility of conducting further experiments to find out whether their synthesis and decomposition are due to exposure to light and time, or are the result of metabolic functions of microbes and algae.

3. It is recommended to add some figures and tables in the Results section, such as records of the experimental process or micrographs of the microbes and algae. This will enrich the experimental content and enhance the credibility of the results.

As an additional material one can add pictures of the enrichment culture.

The enrichment culture after 1.5 months of cultivation

4. Adding subheadings within the Results section for better segmentation would improve the overall structure of the article.

Absolutely agree. Added to the manuscript.

Results and discussion

Composition of exogenous metabolites

The similarity assessment

Composition of the algaе-bacterial community

Conclusion Section:

1. Emphasize the significance of ecological and biochemical mechanisms: In the conclusion, emphasize the significance of these findings in the context of ecological and biochemical mechanisms in freshwater ecosystems.

The results obtained indicate the importance of ecological and biochemical mechanisms in freshwater ecosystems, which are governed by a variety of exogenous metabolites of populations enteringalgo-bacterial communities.

2. Clearly state future research directions: Explicitly mention the potential areas of further research in this field within the conclusion.

Further research should be aimed, among other things, at finding and identifying groups of compounds that could provide, for example, a synergistic effect in suppressing a particular group or species of microorganisms and algae,e.g. cyanobacteria.

3. Discuss the potential practical implications: The conclusion should encompass the potential practical implications of these findings. How can this research provide valuable information for environmental management, water quality control, or other practical applications?

These functions can be practically significant for environmental management and water quality control. In particular, by identifying the most active allelochemics against cyanobacteria, it is possible to further use them as natural algicides to control such a hazardous phenomenon as cyanobacterial "flowering".

In addition, the manuscript's References format requires careful revision.

References edited.

Author Response File: Author Response.docx

Reviewer 2 Report

This is a very meaningful study, and the interactions between bacteria and algae, as well as the interactions mediated by metabolites, are both worth studying. However, this article lacks necessary data support, and if further supporting data can be supplemented, it will be well worth publishing.

Line 115, How to maintain natural light in indoor cultivation experiments, the light intensity will vary greatly, and the influence of light is also very important.

Line 117, cm3, Incorrect unit format. Please carefully check the unit format to meet the requirements of water.

Lines 115-120, Please provide a detailed method process.

Line 121, “The article” should be “this study”.

Results, Lack of necessary physiological parameters to characterize sample status, such as growth curve, biomass, diversity, etc.

Table 1, How to ensure the accuracy of compound identification? And the quantification of compounds, can these represent the quantity and diversity of metabolites?

The role of metabolites in algal-bacterial interactions lacks necessary data support.

no

Author Response

Comments and Suggestions for Authors

This is a very meaningful study, and the interactions between bacteria and algae, as well as the interactions mediated by metabolites, are both worth studying. However, this article lacks necessary data support, and if further supporting data can be supplemented, it will be well worth publishing.

We would like to thank the reviewer for comments.

1. Line 115, How to maintain natural light in indoor cultivation experiments, the light intensity will vary greatly, and the influence of light is also very important.

There were attempts to bringthe experiment closer to natural conditions, so it was madein natural light. The experiment lasted 2 years. The illumination varied depending on the season but it was done on purpose. The article deals with results obtained during the first 1.5 months.

2. Line 117, cm³, Incorrect unit format. Please carefully check the unit format to meet the requirements of water.

Cm³ was replaced by ml

3. Lines 115-120, Please provide a detailed method process.

The experimental enrichment culture obtained on the basis of sampled river water ( the Akhtuba river, the Volga river branch, Astrakhan region, Russia, September, 2009)  was cultured in the nutrient medium BG_N11 [16] for cyanobacteria. The percentage ratio of the river water and nutrient medium when setting up the experiment was 75% to 25%, respectively. We chose this ratio of the water andmedium to intensify the development of cyanobacteria and algae in the presence of the nutrient medium. Cultivation was carried out at 22-25 ° C in natural light. To isolate exometabolites, 250 ml of the culture medium together with cells were filtered through a nylon filter with a pore size of 10-50 µm. The resulting culture fluid was then placed in a 250 ml separation funnel, 3 ml of hexane was added and gently shaken for 3 minutes, inverting the funnel. After 5 min settling, the fluid was drained, and the hexane that appeared on the surface was drained separately into a glass bottle with a stopper. The extracts were kept in the freezer at -18°C before conducting GC-MS analysis. When extracted with nonpolar solvents (hexane, petroleum ether, gasoline, etc.), a large group of biologically active substances is released by plants and algae [17]. To replanish the utilized culture medium, 250 ml of BGN11 medium was added every two weeks.

4. Line 121, “The article” should be “this study”.

We agree. It’s been revised.

5. Results, Lack of necessary physiological parameters to characterize sample status, such as growth curve, biomass, diversity, etc.

We agree. A growth curve would be illustrative. But we were quantifying bacteria and fungi, which is tabulated. We also evaluated the diversity of algae and cyanobacteria as  described in the paper. Every two weeks, 250 ml was taken to prepare hexane extract to determine the chemical composition of secondary metabolites. Algae and cyanobacteria developed unevenly in the cylinder, fouling the container walls, bottom, and surface. Biomass was not determined because scraping  phototrophs off the walls every two weeks could affect further growth of the culture.

6. Table 1, How to ensure the accuracy of compound identification? And the quantification of compounds, can these represent the quantity and diversity of metabolites?

LMWOCs were identified using NIST-2008 and Wiley mass spectra libraries. For more accurate identification, linear retention indices [18] obtained using C7 - C30 alkane standards were used. The accuracy of the identification of the compounds given in the article was ensured by taking into account the Match and Reverse Match factors (R.Match), which were at least 800-900 (good match). In many cases for the most abundant compounds they were > 900 (excellent match). Quantitative analysis was performed using benzophenone as an internal standard. This analysis allowed us to determine exactly the content of the identified compounds. The diversity of the compounds was determined by the qualitative analysis, namelyin the process of compound identification.

7. The role of metabolites in algal-bacterial interactions lacks necessary data support.

The authors agree that data showing direct interactions by means of exometabolites between components of algal-bacterial communities were not obtained in this work. However, we have indirect evidence of such interactions, namely, qualitative and quantitative changes in the composition of LMWOCs with simultaneous changes in the composition and number of microorganisms - members of the algal-bacterial system. In addition, we provide literature data on the functional role of certain LMWOCs in aquatic ecosystems. With a high degree of probability, we can assume that in our studied enrichment culture the metabolites appearing inside the system perform similar biochemical functions, ensuring the interaction amid components of the algal-bacterial community. Allelopathic interactions, as it seems to us, are one of the most important in the joint successional development of microorganisms and algae inside the enrichment culture community.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thank you very much for the author's response and modifications. I have no further comments and agree to publish.