Dear Michael and co-authors

Your manuscript has provoked three very critical reviews but all reviewers agree that you also generated highly interesting results on growth and stable carbon/oxygen isotope fractionation in the calciumcarbonate of four different coccolithophore species. The main problem the reviewers have with your manuscript is related to the core parameter of your study (DCUt). To calculate this parameter you assume that “the passive influx of CO2 constitutes the only source of carbon to the cell” (i.e. for particulate organic and inorganic carbon (POC and PIC, respectively)). This statement (and I concur with the reviewers) is not correct.

I have not followed all the literature on “carbon uptake” into coccolithophorids, but a major portion of it was generated by our group at AWI. More than 10 years ago Rost et al. (2002) have shown that carbon isotope fractionation in Emiliania huxleyi cannot be explained by diffusive fluxes, i.e. CO2, alone. Using a MIMS (Membrane Inlet Mass Spectrometer), Rost et al. (2006) and Trimborn et al. (2007) have demonstrated the presence of a CCM (Carbon Concentrating Mechanism) that also uses bicarbonate as a carbon source (especially at low pCO2). Using the 14C disequilibrium method, Rokitta and Rost (2012), confirmed the uptake of bicarbonate ions. Because, their assay is carried out at pH 8.5 the amount of bi-carbonate used as a carbon source may have been over-estimated. However, by now, bicarbonate transporters as well as their up and down regulation in dependence to carbonate chemistry (notably pH) have also been demonstrated in coccolithophorids, (Mackinder et al., 2010; Rokitta and Rost, 2012). Just recently, Kottmeier et al. (2014) used a modified 14C disequilibrium method (at ecologically relevant pH) and demonstrated a strong shift from bicarbonate to CO2 uptake in Emiliania huxleyi with acidification (i.e. at higher CO2 concentrations).

The bottom line is that bicarbonate seems to be a substantial fraction of the carbon taken up in both PIC and POC. In your response to reviewer 1 you write: “We will explain more carefully and justify the use of aqueous CO2 to represent the supply of DIC from the external milieu into the cell as a whole, or to best approximate it, and why we can conform to the authoritative empirical work by Bidigare et al. (1997)”. My question then becomes, do you not believe the evidence in the above mentioned papers demonstrating the uptake of bicarbonate? If you really think their conclusions are wrong, you should clearly state why! However, in my view, the Bidigare et al., (1997) paper has been proven wrong and therefore is senseless as a focal point for your paper.

In your reply to reviewer 2 you state: “we feel that any differences between mechanisms and routes of carbon assimilation and acquisition by coccolithophores are minor and make very little difference to the conclusions and the palaeoceanographic implications of the present study. We would like to reemphasise that the phenomenon of diminished vital effects at high carbon availability is the primary conclusion of this paper.”
Although, I do not agree with the first part of this statement (that the differences in mechanisms and routes of carbon assimilation and acquisition are minor and make very little difference to the conclusions. On the contrary, they are principally very different), I do agree with you that the phenomenon of diminished vital effects at high carbon availability is the primary conclusion of your paper (I will add though that “vital effects” are probably still active but that the outcome is not “visible”).

In your reply to reviewer 2, you suggest 3 options for a revised manuscript. I encourage you to resubmit a revised version using option 3 (Completely remove the DCUT index (sections 3.3. and 4.2.), and focus on a qualitative interpretation of isotopes and paleoceanographic outlooks only. Please note that this also requires you to rework section 4.3). Please use the reviewers comments and remarks to prepare the final (BG) manuscript, taking also into account what I have stated above.

In the revised manuscript please state explicitly which parameters of the carbonate system were used to calculate it, how it was calculated and present the full carbonate chemistry in a table (along with other data, such as salinity, pH scale, omega, number of replicates, etc.)

As argued by the first reviewer, omega values are very high at the high DIC end of your treatments. Did you see inorganic CaCO3 precipitation. If corrected for PIC and POC (were those measured?) does that leave room for inorganic precipitation?

Literature:
Kottmeier, D.M., Rokitta, S.D., Tortell, P.D. and Rost, B. (2014) Strong shift from HCO3 (-) to CO 2 uptake in Emiliania huxleyi with acidification: new approach unravels acclimation versus short-term pH effects. Photosynth Res 121, 265-275.
Mackinder, L., Wheeler, G., Schroeder, D., Riebesell, U. and Brownlee, C. (2010) Molecular Mechanisms Underlying Calcification in Coccolithophores. Geomicrobiology Journal 27, 585-595.
Rokitta, S.D. and Rost, B. (2012) Effects of CO2 and their modulation by light in the life-cycle stages of the coccolithophore Emiliania huxleyi. Limnology and Oceanography 57, 607-618.
Rost, B., Riebesell, U. and Sültemeyer, D. (2006) Carbon acquisition of marine phytoplankton: Effect of photoperiod length. Limnology and Oceanography 51, 12-20.
Rost, B., Zondervan, I. and Riebesell, U. (2002) Light-dependent carbon isotope fractionation in the coccolithophorid Emiliania huxleyi. Limnology and Oceanography 47, 120-128.
Trimborn, S., Langer, G. and Rost, B. (2007) Effect of varying calcium concentrations and light intensities on calcification and photosynthesis in Emiliania huxleyi. Limnology and Oceanography 52, 2285-2293.

Dear Michael and co-authors,

Congratulations to a fine piece of work. You have substantially rewritten the BGD version and addressed almost all issues raised by the three reviewers. There is one bigger issue (# 13 below) and only a few minor issues that need to be dealt with before final publication in BG:

1) In the summary and later in the text, e.g. p. 5, line 15 you refer to present-day DIC or pCO2 levels. On p. 7 (line13) you write: “under present-day CO2 levels (~ 270 ppm / ~ 2 mmol DIC) is….”. But that is the pre-industrial pCO2 value! On p. 7 (line 2) you write: "Cell division rates at present-day (pre-industrial) DIC levels….”. You refer to pre-industrial values in all cases. Please change present-day to pre-industrial throughout the text. Note, however that present-day should stay on p. 3, line 26:” due to its dominance in present-day oceans, ….”

2) On p. 3 (line 26) you mention pH for the first time: ”… composition under a wide range of DIC (hence pCO2) levels at constant pH (8.2) recreated in the…..”. Please add "total scale".

3) Salinity has no units: p. 4, line 28/29: “….The batch of seawater (salinity ~ 33 psu)….” Change to (salinity ~ 33). Also in Table 1.

4) On p. 4, line 33, you mention the “K/2 recipe”. Is it really K/2 (Keller, 1984) or did you mean F/2?

5) When you show a formula, e.g. p.5 line 30, please include the units of the parameters in the text above or below the formula, e.g. p. 5, line 26/27: “The specific growth rates (μ; day-1) were calculated from cell densities measured at time of culture harvest (cf; cells/ml) and two…..” Please do this for all your formulas where required.

6) P. 8, line 25/26 you write: “One may expect decreased μ to be accompanied by longer generation time, and hence larger cell sizes (Aloisi, 2015).” Isn’t “decreased µ” the same as “longer generation time”?.... and should this necessarily lead to the conclusion that cell sizes become larger? I’m just wondering.

7) Please check all your units in the text and in the table! E.g. p.6, line 34: “3.1 Modern-day dissolved inorganic carbon concentration (~ 2 mmol DIC)”. This should be “3.1 Modern-day dissolved inorganic carbon concentration (~ 2 mmol/kg DIC)” or “3.1 Modern-day dissolved inorganic carbon concentration (~ 2 mmol kg-1 DIC)”. I leave it up to you if you want to use “/kg” or “kg-1”.
Page 7, line 2: “Cell division rates at present-day (pre-industrial) DIC levels (~ 2 mmol) are similar to….”. This should be “Cell division rates at present-day or pre-industrial DIC levels (~ 2 mmol/kg) are similar to….”. On the same page, line 9/10: “species at 2 mmol DIC,……”. This should be “species at 2 mmol/kg DIC,……”. Same page, line 13/14: “under present-day CO2 levels (~ 270 ppm / ~ 2 mmol DIC) is considerable,….”. This should be: “under pre-industrial CO2 levels (~ 270 ppm / ~ 2 mmol/kg DIC) is considerable,….”.
Some more examples: Page 9, line 2:” …..values at 2 mmol DIC show a….”. should be: “….values at 2 mmol/kg DIC show a……….” or “……….values at 2 mmol kg-1 DIC show a….”. Same page, line 8: “…..lowest δ13C at 2 mmol….”. Should be “…..lowest δ13C at 2 mmol/kg….”. Same page, line 26: “…..12 mmol of DIC, yet…..”. Should be ““…..12 mmol/kg of DIC, yet…..””….etc.
Please check this in the entire text. Also, please note that µM is µmol/l and NOT the same as µmol/kg. Please replace µM with µmol/kg. In table 1 you write “µM kg-1”. As far as I know that would be equivalent to µmol l-1 kg-1, which is not correct. Please change to “µmol kg-1”.

8) On p. 10, line 5-7 you refer to Kottmeier et al., 2014: “Likewise, we cannot explain the isotopic data of coccoliths by a shift in the relative assimilation of HCO3- and CO2 by the cells with changing ambient DIC concentration (Kottmeier et al., 2014).” However, this reference is out of place as you are talking about the PIC fraction while Kottmeier et al., 2014 refer to POC!!!

9) On page 11, line 1 all of a sudden the term Ci shows up (…..may imprint the whole Ci leading to substantial isotopic consequences….). Please explain or introduce this term first.

10) Page 11, line 6/7 you write: “…..(for reference: ε13CO2 aq –HCO3- ~ 10 ‰ and ε13calcite–HCO3- ~ 1 ‰ at 15 °C; Zeebe and Wolf-Gladrow, 2001).”. Since you are referring to Fig. 2a, it might be more useful to use delta values instead: In Fig. 2a the reference d13CDIC is 0 (as your inorganic reference d13CCalcite is 1); Then d13C_HCO3 is ca. 0 and d13C_CO2aq is ca. -9 permil. I suggest to use those numbers.

11) You use terms like “isotopically higher than….” (p. 11, line 6), “high δ13C” (p. 11, line 13), “very light δ13C values” (p. 11, line 25) etc. The mixed usage of high vs. low, heavy vs light, or enriched vs. depleted throughout the text can be misleading as e.g. low values may be enriched, etc. I suggest to stick to one set of terms.

12) On p. 13, line 3 you write: “Comparing our isotopic data for….”. Data are not isotopic; you mean “isotope data”.

13) Last but not least (!). In fact, my last comment should go on top. Good scientific practice requires you to place your new data in the framework of existing data (and I mentioned some references in my response at the end of the discussion phase and in preparation for the final msc). There are published data for E.hux on d13C_PIC as a function of light intensity and [CO2] (Rost et al., 2002) that I suggest you plot those data in Fig 2a. Take the ones at photon flux densities of 150 mmol m-2 s-1 (Rost et al., 2002, table 1 (p. 123) the bottom 5 lines:
[CO2] d13Cc-d13CDIC
(µmol/L) (permil V-PDB)
27.2 -1.87
21.3 -1.21
18.4 -0.58
12.4 -0.91
5.6 -0.22

If you plot these data (please verify them again) you will see, 1) the slope is similar to the one you found for E.hux but, 2) it falls below your line of inorganic calcite and therefore the “vital effects” increase with increasing DIC. This is opposite of your conclusion. I have no explanation for this, but you HAVE to mention this to make your article scientifically sound. That means that you will have to state in your discussion somewhere that there are also conflicting data that cannot be explained at this stage, but that require careful investigation in the future. Or you must explain why these data are not scientifically sound.

there are only two minor issues left:

P.4 line 31: psu is crossed out but it is still there. Please remove

P. 12, line 4: “These differences can be tentatively assign to divergences…..“ should be assigned.