the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Properties of exopolymeric substances (EPS) produced during cyanobacterial growth: potential role in whiting events
Marlisa Martinho de Brito
Irina Bundeleva
Frédéric Marin
Emmanuelle Vennin
Annick Wilmotte
Laurent Plasseraud
Pieter T. Visscher
Abstract. Extracellular polymeric substances (EPS) are an important organic carbon reservoir in many pelagic and benthic environments. The production of EPS is intimately associated with the growth of phyto- and picoplankton. EPS plays a critical role in carbonate precipitation through the binding of cations and by acting as a nucleation site for minerals. Large-scale episodes of fine-grained calcium carbonate precipitation in the water column (whiting events) have been linked to cyanobacterial blooms, including of Synechococcus spp.,. The mechanisms that trigger these precipitation events are still debated. We pose that the cyanobacterial EPS, produced during exponential and stationary growth phases plays a critical role in the formation of whitings. The aim of this study was to investigate the production of EPS during a two-month cyanobacterial growth, mimicking a bloom. We further evaluated the potential role of EPS in carbonate precipitation. The production and properties of EPS produced at different Synechococcus spp. growth stages were investigated and carbonate mineral formation within these EPS matrices was determined in forced precipitation experiments. EPS produced during the early and late stationary phase contained a larger amount of negatively charged groups than present in EPS produced during the exponential phase. Consequently, a higher Ca2+ binding affinity of the stationary phase-EPS led to the formation of a larger amount of smaller carbonate minerals (<50 µm) compared to crystals formed in exponential phase-EPS, which were less and larger (> 50 µm). These findings were used to establish a conceptual model for picoplankton bloom-mediated CaCO3 precipitation that can explain the role of EPS in whitings (see graphical abstract).
- Preprint
(2458 KB) -
Supplement
(307 KB) - BibTeX
- EndNote
Marlisa Martinho de Brito et al.
Status: final response (author comments only)
-
RC1: 'Comment on bg-2023-51', Anonymous Referee #1, 25 Apr 2023
The authors conducted a culture-based study and used a complementary arsenal of carefully done measurements to verify their results and observations, and infer how the precipitation of CaCO3 in pelagic cyanobacterial blooms may occur (during whiting events). FTIR, pH drift assays, EPS compositional analyses, etc. They were able to show that the precipitation of CaCO3 (calcite, and to a lesser extent vaterite) coincided with the magnitude of EPS production and the available functional groups on the EPS occurring in early stationary phase cultures. Larger precips were formed during exponential phase, and smaller, more abundant precips were formed during stationary phase.
The larger precipitates early on (Exponential phase cultures) and smaller precipitates observed in later stationary phase is somewhat puzzling. But interpretations were made that help to explain these outcomes, especially when considering natural bloom systems.
The “pH of cultures was around 10, and remained steady”. Given that cultures were grown under 12/12 light/dark cycles, was pH measured in darkness? It should be clarified if pH was measured during light conditions or dark, or both. Please clarify?
Figure 1. What may have caused the dip in both pH (B), and numbers of cells (A) during stationary phase (near day 40) in Experiment 1? Any suggestions?
Using FTIR, highest protein levels (line 256, 257) were indicated, and later using colorimetric assays of protein (line 271) it is stated that highest protein occurred in EPS also during early stationary phase (also shown in Table 3) – good verification!
The FTIR results for EPS are especially informative and helpful. The authors should consider summarizing these in a separate Table for easier reference by the reader.
Although this is a laboratory based study, it sheds light on a longer standing issue of how whiting events occur during blooms in natural systems. The authors are to be commended on the nice, careful work examining this whiting-related process. The ms was well written, and only minor changes are suggested.
Citation: https://doi.org/10.5194/bg-2023-51-RC1 -
AC1: 'Reply on RC1', Marlisa Martinho De Brito, 30 May 2023
Dear Referee,
Thank you for reviewing our manuscript, entitled: "Properties of exopolymeric substances (EPS) produced during cyanobacterial growth: potential role in whiting events" and for providing valuable feedback. We have made revisions to the main text to address each of your points. A detailed response to each one of your comments is provided in the attached document (Referee1_Rebuttal_RM_30May2023), where you will also find the manuscript highlighted with track changes.
Once again, thank you for the time and effort invested in reviewing our work. We believe that your insights have significantly contributed to improving the manuscript.
We look forward to hearing your feedback on the revised manuscript and please let us know if there are any further adjustments or revisions you would like us to consider.Sincerely,
Marlisa Martinho de Brito
-
AC1: 'Reply on RC1', Marlisa Martinho De Brito, 30 May 2023
-
RC2: 'Comment on bg-2023-51', Sven Tobias-Hunefeld, 26 Apr 2023
Summary:
The authors investigated the role of EPS in carbonate precipitation during a Synechococcus bloom experiment. EPS characteristics were assessed with FT-IR spectroscopy, SDS-PAGE and forced carbonate mineral precipitation experiments. The authors separated the bloom into exponential, and early and late exponential stages. Stationary phases contained higher abundances of negatively charged groups than during the exponential phase, leading to smaller (<50um) but more abundant carbonate minerals in the stationary phases, whereas the exponential phase contained larger (>50um) but less abundant carbonate percipitates.I believe this manuscript gives a small but important contribution to the field. I especially applaud the many different approaches taken to measure the EPS and associated factors. My comments mainly centre around cutting down on the amount of references, only using the key ones, and ensuring that the different sections (introduction/results/discussion) don't intersect with each other. Other comments include ways to convey data reliability and acknowledging limitations.
Major comments:
The abstract should make clearer what techniques were used for measurements, at least the ones that the major findings were drawn upon.
The graphical abstract is well made and explains the stated findings well. However, I am unsure of what the 'Negatively charged groups' are, is it the connecting lines of the different phases? In that case I would remove the Exponential/Early stationary/Late stationary illustrations and explanations from the Legend.
There are many references throughout the introduction and discussion, to the degree that the text in the intro is 50% references and 50% informative text. I would focus more on relevant and landmark references and use these key references to make your point. Rather than having 3-5 references for every statement. For example: L83-84: There are 3 references that say that cyanobacteria are known EPS producers, and then another 2 that state this is especially true during blooms. It would be best to have only those last two as they are the most relevant to your point.
Methods seem to match the stated goals of the study. Many measurements however were done in duplicate, with no justification as to why. Such as protein, sugar and glycosaminoglycan quantifications. Unless measurements were extremely similar, triplicates would convey a greater degree of measurement precision. I would recommend either measuring an additional sample (if available), stating the reason for why duplicates were chosen for this, or acknowledging this as a study limitation.
As there were two experimental runs that were temporally displaced, I would have liked to see a brief comparison of their measurements to show that these two test runs are not significantly different. Additionally, is there an explanation why experiment 1 had a 7 day and experiment 2 had a 14 day exponential phase? With the difference in exponential phase why were the chosen sample days the same for both experiments? Specificallly: D0, D14, D8, D56.
By picking only crystals above 10 µm, could this be introducing some bias? Or was the 10µm limit chosen due to methodological limitations? If yes, should be stated. Otherwise, the 10µm size cut off should be justified.
Please add confidence intervals/standard deviations to Figure 5. I would also like to see the statistics represented on all relevant figures. Between negative controls and measured values, as well as between time points.
Please increase the readability of introduction and discussion, lots of results were included and some points are made and justified either in the next discussion section or 20 lines below. Based on this there were some other points to make:
- As EPS characteristics are phytoplankton/organism specific it should be noted that these findings need to be expanded on with additional studies and other phytoplankton.
- Tables and Figures that were already presented in the results section should not be mentioned again in the discussion.
- The same is true for the discussion section, do not reiterate results in this section, but rather focus only on the implications and what this means for the field.
- Ensure that conclusion drawn from the data are found only in the discussion, and not in the results. Such as L276, L278-279.
- Please ensure that you do not discuss or provide justification for the chosen methods in your results, but rather in the introduction, methods section, or discussion (as relevant).
- Please make clear how discussion L381-399 relates to whiting events. The text is very information dense, however how it relates to phytoplankton associated whiting events is not clearly stated. I believe it would help to have L400-417 before L381-399, or remove L381-399 from the manuscript.
As EPS characteristics are phytoplankton/organism specific it should be noted that these findings need to be expanded on with additional studies and other phytoplankton.
Minor comments (please also see attached pdf with comments):
- The limit of detection was stated in µg.L-1 in text, however Table 1 values are in µM. The same units should be
used throughout the manuscript. - I would also be interested in day 0 values throughout the text, as it was stated that they were measured but are not shown in the manuscript.
- Only Figure 2 is required. Table 2 should then be relegated to the supplementaries.
- FT-IR mentions a lot of different spectra, it would be best to only focus on the ones from the discussion or those relevant to the findings.
- Can Figure 4 be redone with the inclusion of a negative control?
-
AC2: 'Reply on RC2, Dr. Sven Tobias-Hunefeld', Marlisa Martinho De Brito, 30 May 2023
Dear Dr. Sven Tobias-Hunefeld,
Thank you for taking the time to review our manuscript titled: "Properties of exopolymeric substances (EPS) produced during cyanobacterial growth: potential role in whiting events". We truly appreciate your rigorous examination of our work and the constructive comments you have shared.
We have carefully revised the main text to specifically address both the major and minor points you raised. To provide you with a comprehensive reply, we have included a pdf document (Referee 2_Rebuttal_RM_30May2023) where you will find a response to each of your comments and the revised manuscript with track changes. Within this document, you will easily identify the modifications we implemented as they are clearly highlighted (in yellow).
Once again, thank you for your valuable feedback. We believe that your revisions have significantly improved the quality and clarity of our manuscript.
We look forward to hearing your feedback on the revised MS.Please let us know if there are any further adjustments or revisions you would like us to consider.Sincerely,Marlisa Martinho de Brito- AC3: 'Reply on AC2, Dr. Sven Tobias-Hunefeld', Marlisa Martinho De Brito, 30 May 2023
- AC4: 'Reply on RC2, Dr. Sven Tobias-Hunefeld', Marlisa Martinho De Brito, 30 May 2023
Marlisa Martinho de Brito et al.
Marlisa Martinho de Brito et al.
Viewed
| HTML | XML | Total | Supplement | BibTeX | EndNote | |
|---|---|---|---|---|---|---|
| 235 | 52 | 12 | 299 | 27 | 4 | 7 |
- HTML: 235
- PDF: 52
- XML: 12
- Total: 299
- Supplement: 27
- BibTeX: 4
- EndNote: 7
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1