Alterations in microbial community composition with increasing <i>f</i>CO<sub>2</sub>: a mesocosm study in the eastern Baltic Sea

Crawfurd, Katharine J.; Alvarez-Fernandez, Santiago; Mojica, Kristina D. A.; Riebesell, Ulf; Brussaard, Corina P. D.

doi:https://doi.org/10.5194/bg-14-3831-2017

Articles | Volume 14, issue 16

https://doi.org/10.5194/bg-14-3831-2017

© Author(s) 2017. This work is distributed under
the Creative Commons Attribution 3.0 License.

Special issue:

Effects of rising CO₂ on a Baltic Sea plankton...

https://doi.org/10.5194/bg-14-3831-2017

© Author(s) 2017. This work is distributed under
the Creative Commons Attribution 3.0 License.

Articles | Volume 14, issue 16

Research article

|

29 Aug 2017

Research article |

| 29 Aug 2017

Alterations in microbial community composition with increasing fCO₂: a mesocosm study in the eastern Baltic Sea

Katharine J. Crawfurd, Santiago Alvarez-Fernandez, Kristina D. A. Mojica, Ulf Riebesell, and Corina P. D. Brussaard

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Final revised paper (published on 29 Aug 2017)
Supplement to the final revised paper
Preprint (discussion started on 02 Feb 2016)
Supplement to the preprint

Interactive discussion

Status: closed

AC: Author comment | RC: Referee comment | SC: Short comment | EC: Editor comment

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- Supplement

RC1: 'Review of Crawfurd et al.', Anonymous Referee #1, 03 Apr 2016
- AC2: 'Reply to referee 1', K. Crawfurd, 20 Jun 2016
RC2: 'Referee comment on „Shifts in the microbial community in the Baltic Sea with increasing CO2“', Anonymous Referee #2, 19 May 2016
- AC1: 'Reply to referee 2', K. Crawfurd, 20 Jun 2016

Peer-review completion

AR: Author's response | RR: Referee report | ED: Editor decision

ED: Reconsider after major revisions (20 Jun 2016) by E.P. Achterberg

AR by K. Crawfurd on behalf of the Authors (02 Aug 2016) Author's response Manuscript

ED: Referee Nomination & Report Request started (18 Sep 2016) by E.P. Achterberg

RR by Anonymous Referee #2 (16 Oct 2016)

RR by Anonymous Referee #1 (18 Oct 2016)

Suggestions for revision or reasons for rejection

As I also reviewed the original submission, I am omitting a summary of the manuscript here.
Although the authors changed the title of their manuscript, the interpretation of the presented data hasn’t changed much (or at least it doesn’t come across to the reader). At the moment it is relatively hard to judge the presented manuscript/data as there are still many cases where the statements/conclusions/claims by the authors are not supported by the results. Unfortunately, the authors do not present any further analyses or statistics to back up their statements (I have given a few examples below; however, this list is not comprehensive!). An overall suggestion to the authors would be to look for someone who would be able and willing to aid with the manuscript re-structuring and re-writing, but who is himself or herself not involved in these experiments (someone in their departments maybe? I think it would help to get an outside-the-box opinion here for the interpretation of the data). At the current state, there seem to be too many misinterpretations in the manuscript in order to recommend publication (even with some revisions). I have made some more specific comments below.

In general, the authors try to make (big) claims about the effect of CO2 on the microbial community; however, basically provide neither analyses/statistics that support these claims nor is it visible in the figures. Further, the authors have not given any type of error on their measurements making it hard to see actual differences. Overall, in many places it seems that the authors see what they want to see (i.e. an effect of CO2 concentrations on the microbial community).

Again, I would recommend to add an overview of the experimental setup to the methods section. I understand that the experimental setup is described in a different manuscript for the special issue. However, as a reader of this manuscript, I actually do not want to have to read another manuscript first in order to find out what the authors did here; at least a summary would be needed here.

Many places in the manuscript are still not written to the extent that they can be easily followed, for example: “The higher loss rates (days 5 to 9; Fig. 3e) resulted in a decrease in abundance, which was stronger for the low fCO2 mesocosms (as illustrated by M1) due to the significantly higher gross growth rates for the high fCO2 mesocosm (represented by M3; Fig. 3b). The positive correlation of Pico I peak abundance with fCO2 on day 13 (Fig. 3h, R2=0.94) was lost upon another decline in abundance. Significantly higher losses at high fCO2, a combination of grazing and lysis, resulted in a more dramatic crash at high fCO2 and abundances becoming similar again around day 17 (Fig. 3a).” This paragraph is simply not understandable for someone not working on these experiments without having to read the sentences multiple times (which disrupts the flow reading dramatically).

The figures were not labeled at the end of the manuscript (no matter whether this is because of forgetting to label them or whether the submission software omitted to do so); so, for the purpose of the review I assumed that the figures were 1-11 in the way they came out of the printer and I am referring to them in that way.

In general, I think that the authors should try to improve the readability of their manuscript by increasing the quality of their figures, e.g., not having figures that span nearly two full pages (see Figure 3) or omitting repetitive data.

Showing the ambient data together with the mesocosm is essential to judge whether changes in the mesocosms were, for example, induced by enclosement or were naturally happening outside as well. This does not mean that, in case changes were induced by enclosement, that differences between mesocosms are not useful; they are valuable data. Not presenting the ambient microbial community leaves the reader with guessing what happened and potentially suspecting that the data was omitted because they would alter the conclusions of the paper. If the authors would like to increase the understandability of their manuscript, I highly recommend adding the ambient data to the current main manuscript figures.
For example, Suppl. Figure 1 shows that changes in the Synechococcus population are pretty much the result of ‘bottle effects’ (I agree that a mesocosm is a large bottle, but it is an enclosement). Is this possibly related to cleaning procedures or stirring of the mesocosms as outlined in Paul et al.? While other groups are coherent with the ambient water.

After looking into Paul et al. for the experimental setup, it seemed that the CO2 concentrations were actually measured during the mesocosms, why aren’t the actual measured concentrations used for any of the plots instead of the targeted concentrations (which were only achieved in the first few days but weren’t maintained)? This seems such an obvious thing to do and a fact that is completely ignored by the authors.

Abstract: “Of these groups 2 picoeukaryotic groups increased in abundance whilst the other groups, including prokaryotic Synechococcus spp., decreased with increasing fCO2.” Looking at both Figure 2a and Suppl. Figure 1a, I seriously do not see where this statement comes from! There is a short period of time (~ 7-16 days) where Syn. are lower in the low fCO2 than in the higher ones but for most of the experiment it is the other way around. Unfortunately, the authors also do not supply any other analysis or statistics that could possibly back up their claim. Unfortunately, these kind of statements and interpretation is occurring throughout the manuscript, which leads me to the suggestion that the current manuscript is far from being acceptable for publication at this time.

Figure 1: The authors claim that the decline in total phytoplankton around the end of phase 2 and throughout phase 3 is due to a decrease in Synechococcus. This is supported by Figure 2a. However, Suppl Figure 1a shows Synechococcus populations staying up rather than declining to concentrations in Figure 1. After doing some research through this manuscript, I found that the difference between the figures is the depth reference here, which is 0.3-10 m for Figure 1 and 0-17 m for Suppl. Figure 1. This difference actually suggests that either the distribution of Synechococcus is not equal throughout the water column or that there is a sinking out of Synechococcus? I don’t know why this is, maybe the authors should think about this deviation which is currently not acknowledged anywhere.

Figure 1: The authors say that the total phytoplankton abundance at the end of phase 1 is significantly higher in the high fCO2 treatment than the low fCO2 treatments. Again, I am really sorry, but I cannot see this in the plots and the authors again don’t provide any analysis or statistics for this. (see p 10 lines 18-20)

Figure 2: Panel c is absolutely unnecessary here (and in following figures); it is the same data as in panel a and its addition here suggests that it is indeed different data and is making the figure unnecessarily large. Panel d: What are the errors on these measurements? I wonder whether these differences are actual changes or just variation? Further, this is comparing growth rates from time point 10 days to growth and loss rates of time points 4-7. In my opinion, this is totally misleading as day 10 is obviously different than 4-7 as can be seen in panel a. (see p 11, l 10-13).
Figure 2a legend: It is not necessary to present 4 (!) different significance levels here. For most claims in the manuscript no analyses or statistics are provided while here it is over the top. Either s.th. is significantly different or not.

p 10, l 21 and following: If CO2 has a strong positive effect on Pico 1 and 2, how can they then be comparable in abundances to the surrounding water? Maybe I don’t understand this sentence.

Suppl. Figure 1: This is (probably) the most informative figure of the entire manuscript.

p 11, l3-5: This claim is not supported by any analysis. Any time series statistics, PCA, similarity analysis, community composition comparisons?

p 17, l 17-18: this is an odd comparison in size fractions and the results are not exclusive of each other.

p 18, l 20-22: There is consistency among the mesocosms maybe, but there is already a deviation to the surrounding water indicating an effect of enclosement, at least on some groups.

Figure S3 is missing!

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RR by Anonymous Referee #3 (22 Nov 2016)

Suggestions for revision or reasons for rejection

This study investigates the effect of different fCO2 levels on different microbial groups as determined by flow cytometry in mesococm experiments in the Gulf of Finland. Clear and differential effects of fCO2 levels were found for the various microbial groups. In two mesocosms (low and high fCO2), viral infection and grazing were assessed, parameters that have not been very often measured in ocean acidification experiments.

The experimental approach and the methods are appropriate and appropriately described. Useful information on the potential effect of ocean acidification on different microbial groups was obtained.

The differences between M1 and M3 (growth rates, viral infection and grazing) are a bit overemphasized. Differences between two mesocosms can certainly be calculated and this might be ok in some experimental approaches. But in this case samples have to be taken repeatedly within one mesocosm to estimate the entire variability of the approach. This was certainly not done for the viral infection and grazing measurements. Also, the authors use this analysis to make a comparison between two treatments, but this can also be done when the experimental treatments are replicated. I appreciate the effort that the authors made, but they put to much weight on this analysis. They are right not to mention the effect in the abstract and should reduce the reference to these findings in the discussion. It is ok to discuss that briefly as potential effect, however, in the absence of statistical prove, this part short be less prominent.

Despite the comments of one reviewer, the presentation -especially in the discussion- remains poor. Many sentences are awkward or hard to understand. Also, the arguments are sometimes presented in a sloppy way. Although I think I understand what the authors want to say and have no serious objection, the argumentation has to be improved and presented with more rigor.

Minor comments:

Page 2
Line 5/6: Does this mean that TOTAL algal biomass is related to prokaryotic biomass, despote of differential effects of fCO2 on algal groups? Please specify.

Page 3
Lines 21-23: specify the direction of the responses.

Page 10
Results: I think there should be a short part on phase 0.

Page 11
Line 10: ‘most so’. generally?
Line 19: Are low and high fCO2 mesocosms nowhere defined.
Line 22: What does ‘not so clear’ mean? Please use a more rigorous (scientific language). This comment holds also for many other occasions; not to all will be referred to.

Page 12
Line 2-4: I.e., 26% of total phytoplankton abundances were found on average in the high high fCO2 mescosms? Please use more verbs in your sentences, this would increase the readability of the text. This holds for the entire text.
Line 22: What exactly do you mean by ‘matched’.

Page 13
Lines 6-7: These? Please explain better what you mean.
Line 8: explain better why the word seems is appropriate here.
Line 17 remove just
Line 22: remove indeed
Line 24: remove perfectly
Line 25: remove indeed

Page 14
Line 2: largely - be more precise
Line 3 after r’easons’, add: see materials and methods
Lines 4-7: Awkward sentence
Line 23: Dynamics is I think a word that requires the singular.
Line 23-25: No idea what the authors mean.

Page 15
Line 7: change into to to
Line 2-3: A correlation could be calculated

Page 16
Lines 2-3: give test and significance level

Page 16/17
There was only one mesocosm for low fCO2 level, right?

Discussion
1st paragraph: There is no real explanation given. Only net growth and loss rates are observed. What has been actually ‘examined'?
2nd paragraph: Why would only nano-sized and not other phytoplankton profit from the nutrient upwelling? Also, the argumentation is not very clear.

Page 19
1st paragraph: ‘difficult’ is sufficient. In addition, the authors could just refer to the pigment analysis. The molecular analyses were not done, so there is no need to refer to them. Again, no explanation is given, just rates are listed. The reader has to try to figure out what the authors have calculated. This has to be explained better.
2nd paragraph: what are ‘relatively’ high loss? How can, based on net changes of abundances and some loss rates estimates, turnover of organic matter be calculateds.

Following pages
Same problems as with other parts of the discussion. Please spend more time on a better and especially more rigorous explanation and argumentation! It is necessary that one can follow your arguments in the text!

Figures: The legend starts should start with a cation that specifies the intention of the graph.

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ED: Reconsider after major revisions (06 Dec 2016) by E.P. Achterberg

AR by K. Crawfurd on behalf of the Authors (01 Jun 2017) Author's response Manuscript

ED: Publish subject to minor revisions (Editor review) (21 Jun 2017) by E.P. Achterberg

The resubmitted manuscript has substantially improved following two rounds of reviews. In particular the substantial and thorough amendments to the manuscript following the latest reviews have greatly improved the manuscript. The text flows well, and the scientific reasoning is well laid out and expressed.
The use of statistical techniques to show the different behaviours with time of the various phytoplankton communities that were subjected to different CO2 treatments has strongly improved the manuscript.
The authors have followed the recommendations by the reviewers and thereby greatly strengthened the manuscript.
I suggest a number of minor amendments to the manuscript before it can be published.

Line 29: replace dissolution with uptake
Line 29: after carbon dioxide write (CO2)
Line 29: replace in with by
Line 30: replace atmosphere with ocean
Line 32: replace highly with strongly
Line 37: replace CO2 with fugacity of CO2 (fCO2)
Line 39: their abundances with its abundance
Line 429: between “shift towards” place with increasing fCO2
Line 50: replace activities with emissions
Line 50: write: primarily caused by the burning……
Line 56: replace productivity with production
Line 81: replace nutrients with nutrient
Line 81: replace concentration with concentration
Line 84: write: carbon sequestration in deep waters and sediments
Line 101: replace under with with
Line 101: write: temperature conditions
Line 117: replace hung with reached
Line 127: replace fCO2 with fugacity of CO2 (fCO2)
Line 134: write: threshold fCO2 level, if present……
Line 136: write: Initial fCO2 was 240……
Line 166: replace concentrations with levels
Line 300 and 302: replace of with in
Line 306: write: phytoplankton abundance
Line 337: write: low fCO2 compared to high fCO2
Line 448: replace on with for
Line 499: is there evidence from other mesocosm experiments of stimulation of SYN due to bubbling or enclosing?
Line 504: replace occur with upwell
Line 660: replace predicted with projected

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AR by K. Crawfurd on behalf of the Authors (24 Jun 2017) Author's response Manuscript

ED: Publish as is (03 Jul 2017) by E.P. Achterberg

AR by K. Crawfurd on behalf of the Authors (11 Jul 2017) Author's response Manuscript

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Short summary

Carbon dioxide (CO₂) is increasing in the atmosphere and oceans. To simulate future conditions we manipulated CO₂ concentrations of natural Baltic seawater in 55 m³ bags in situ. We saw increased growth rates and abundances of the smallest-sized eukaryotic phytoplankton and reduced abundances of other phytoplankton with increased CO₂. Viral and bacterial abundances were also affected. This would lead to more carbon recycling in the surface water and affect marine food webs and the carbon cycle.

Alterations in microbial community composition with increasing fCO2: a mesocosm study in the eastern Baltic Sea

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Alterations in microbial community composition with increasing fCO₂: a mesocosm study in the eastern Baltic Sea