Changes in Free-Living and Particle-Associated Bacterial Communities Depending on the Growth Phases of Marine Green Algae, Tetraselmis suecica
Round 1
Reviewer 1 Report
The subject approached by the research team is of great scientific interest, at the same time having a great practical importance in the field of algal biomass production for a diverse range of applications.
The introductory part is adequately elaborated, the authors arguing the economic importance of their research and the limitations of the methods used so far in evaluating bacterial communities in algal cultures, as well as the interactions between bacteria and algal cells.
The research methodology is detailed, it is presented with accuracy and professionalism and well organized in eloquent subchapters. Also, the experimental results are properly structured and are accompanied by clear, skillfully elaborated figures, thus suggestively completing the statements in the text.
The comments based on the results obtained are very interesting, pertinent, they are written in a logical flow of ideas, the authors also referring to some results from the scientific literature.
The originality of this research is very high; It should be noted that this is the first study aimed at characterizing the FLB and PAB communities in T. Suecica culture, by the next generation sequencing.
Conclusions are clear and concise; the authors propose some new research directions for a more-in-depth assessment of the impact of bacterial communities on T. suecica marine algae.
Author Response
>Reply: We thank you for your warm comments.
Author Response File: 
Author Response.docx
Reviewer 2 Report
See comments in the attached file.
Comments for author File: 
Comments.pdf
Author Response
Thank you for your comments and suggestions. We have revised the manuscript in accordance with your comment. For your convenience, we marked the revised parts in red. Please, see the reivsed manuscript file, and we also uploaded the response to your comments (please see author-coverletter-1056522.v3.pdf).
Author Response File: Author Response.pdf
Reviewer 3 Report
Authors first to characterize FLB and PAB communities in T. suecica culture using NGS approach.
In method, the number of replicates used in this study are missing.
Author didnt describe how they are handled the micro biota present in water, Removal process ? or show evidence the followed method will not impact the results described in MS. Control samples are missing here. i.e, Sea water.
Author Response
Thank you for your comments and suggestions. We have revised the manuscript in accordance with your comment. For your convenience, we marked the revised parts in red. Please, see the revised manuscript file.
- Authors first to characterize FLB and PAB communities in T. suecica culture using NGS approach. In method, the number of replicates used in this study are missing.
>Reply: In our study, unfortunately, there was no replicate. However, our data are thought to be reliable. As you can see figure S1, rarefaction curves are saturated, indicating that our sequencing depth was enough to show bacterial community composition in this study. In addition, we collected samples for FLB and PAB communities in four different growth phases of T. suecica (lag, exponential, stationary, and death phase), and characterized each community using NGS approach. Moreover, we found that the same-staged cultures were not much different in bacterial cells among replicated cultures. Together with these, the data (T. suecica-growth associated bacterial communities) from our study can be reliable even though there was no replicate. I hope this clarifies our reasoning.
- Author didnt describe how they are handled the micro biota present in water, Removal process? or show evidence the followed method will not impact the results described in MS. Control samples are missing here. i.e, Sea water
>Reply: We thank you for your comments. In the present study, we have analyzed bacterial communities in normal algal culture (f/2 media). Prior to inoculation of algal cells into f/2 media, f/2 media is usually autoclaved in order to remove living organisms, including bacteria. Thus, in this study, we think that control (natural seawater) would not be necessary. I hope this clarifies our reasoning. However, we also think your concern is reasonable, and we have revised (Please, see L75).
Reviewer 4 Report
Comments for authors:
The authors studied the differences between free-living and particles-associated bacterial community during different growth phases of a green algae species, Tetraselmis suecicaI. The topic is very interesting because marine heterotrophic bacteria is the main player in the interaction of plankton community and contribute to the global carbon cycling. Different growth phases of phytoplankton could provide distinct niches and substrates for bacteria. By using 16S rDNA sequencing of the samples that they defined, “free-living” and ”particles-associated” bacterial community, they provided what the microbial community of T. suecicaI looks like and some key bacterial players in specific samples.
My main concerns are from two points:
- The cell size of this green algae, Tetraselmis suecicaI, is about (or a little less than)10 μm. The author’s definition of the samples from the filter with 10 μm pore size as particles-associated bacterial community seems problematic as the filter could not remain the algae cells efficiently leading a lot of algal cells exist in “free-living” samples which make the bacteria in this part are not really free-living. It also reflects from the results, high proportion of shared OUTs between the two parts. Whether the authors could add some statements on why they chose this filter size and explanations about these particles in the medium, what is the relationship between the particles and algae cells?
- Another shortcoming of this experiment is the authors did not set biological replicates for their community experiments, which makes the comparison of different growth phases unreliable. whether the authors could justify it for themselves?
Some minor comments:
Page3, paragraph 4, “0.1 g of the environmental DNA template” should not be “0.1 g”
Figure 3. characters A, B and C are missing in figure.
Author Response
Thank you for your comments and suggestions. We have revised the manuscript in accordance with your comment. For your convenience, we marked the revised parts in red. Please, see the revised manuscript file.
1. The cell size of this green algae, Tetraselmis suecicaI, is about (or a little less than)10 μm. The author’s definition of the samples from the filter with 10 μm pore size as particles-associated bacterial community seems problematic as the filter could not remain the algae cells efficiently leading a lot of algal cells exist in “free-living” samples which make the bacteria in this part are not really free-living. It also reflects from the results, high proportion of shared OUTs between the two parts. Whether the authors could add some statements on why they chose this filter size and explanations about these particles in the medium, what is the relationship between the particles and algae cells?
>Reply: We are sorry for confusion. Cell size of T. suecica which we examined is ranged from 11.8 to 19.1 (average 16.2 μm). Thus, 10 um pore size filter is thought to be useful to distinguishably collect FLB and PAB communities. Moreover, if we used smaller pore size filter, some FLB cannot penetrate the filter (10 um, for PAB communities) since T. suecica blocked the pore. Even, T. suecica cells might be easily clogging the small-sized pores. Thus, we thought that 10 pore size is the most suitable for our study.
2. Another shortcoming of this experiment is the authors did not set biological replicates for their community experiments, which makes the comparison of different growth phases unreliable. whether the authors could justify it for themselves?
>Reply: As you mentioned, unfortunately, there was no replicate in our study. However, we think that our data are reliable. As you can see figure S1, rarefaction curves are saturated, indicating that our sequencing depth was enough to show bacterial community composition in this study. In addition, we have collected samples for FLB and PAB communities in four different growth phase of T. suecica (lag, exponential, stationary, and death phase), and characterized each communities using NGS approach. Together with these. The data (T. suecica-growth associated bacterial communities) from our study can be reliable even though there was no replicate. I hope this clarifies our reasoning.
Some minor comments:
`. Page3, paragraph 4, “0.1 g of the environmental DNA template” should not be “0.1 g”
>Reply: This is our careless mistake. We have revised this. Please see L131
2. Figure 3. characters A, B and C are missing in figure.
>Reply: Thank you for your comment. We edited this figure in accordance your comments. Please, see Figure 3.
