Dear Nemiah Ladd and co-authors,

First I would like to thank the reviewers for their extensive review of your manuscript and of course you for your thorough reply. Hydrogen isotope fractionation is a complex topic as the literature has shown over these last few years, even when studying the effect of only one parameter in culture. When you move into environmental settings it becomes even way more complex. I do think your manuscript addresses a lot of these issues, but I do want to share some of my thoughts based on the reviewer comments that might help you with the interpretation and discussion. You plotted your data against temperature and discussed the possible effects of production/productivity and light, but to some degree these three are correlated in multiple ways complicating a straight forward explanation. One possible explanation for hydrogen isotope variation that evolved over these last few years is the origin of the H of NADPH, directly from photosynthesis or the OPP pathway (e.g. Maloney et al., 2016). In a system with less nutrients and high light you could imagine cells to be active but none growing, producing storage compounds and potentially shifting to relatively more OPP derived NADPH over time than cells growing at high light and nutrients. Nutrient limited growth at high light might also result in other ways to get rid of reducing power potentially affecting the hydrogen isotopic composition of the remain NADPH (NADPH-NADH). Although a bit speculative perhaps these types of process could help explain some of your observation.

As for heterotrophy, for the mixotrophic algae these types origin of NADPH changes might be even more obvious. You discuss a heterotrophic signal during winter that disappears during summer. I think the main reason for that is the overwhelming phototrophic signal during summer, not the absence of heterotrophic organisms. Typically heterotrophs follow their food source, the algae bloom(s) and contribute to the more general fatty acid pool. Either their contribution is relatively low or the isotopic composition of their fatty acids is not extremely different form that of the algae? They dominate the signal during winter, but are more abundant during the spring and summer algae blooms. This would of course be more obvious in typical bacterial fatty acids that are difficult to measure since they are present in relatively low abundance, unfortunately.

I was very pleased to read that you are working on phytol isotopes and chlorophyll as productivity marker, I hope this will be possible to do.

As for the cell count issue raised by reviewer 1, why not add one sentence stating why you did not calculate yield per cell. “Cell counts from the same time period but different samples agree with…”. Of course you might be able to play with chlorophyll concentrations as measure for algal productivity to see how this might correlate with production, temperature and lipid isotopic composition.

I would agree with your reluctance of calculating weighted averages of different lipid types, or lipids with potentially different origins. I am not sure which lipids are discussed here. What I can imagine is that a weighted average of all fatty acids is used, for instance, to be able to say something community metabolism or to correlate fatty acid isotopes with water isotopes or other environmental parameters. That way avoiding or averaging out individual species or biosynthetic pathway effects.

Changing old figure 3 to new figure 3 is fine with me, old figure 3 does contain a lot more information and should definitely end up in supplemental material.

The carboys you used are transparent, but I am assuming the system was gastight, did you measure oxygen inside the carboy? I am assuming that 6 hours might not be enough to create serious issues, but be careful.

Not referencing general protocols, I think that referencing a method and indicating the changes or modifications might be a better way to do this. I would definitely include a reference to extraction study or state that the extraction was optimized by testing various temperatures and time periods that that this was optimal.

The effect of light intensity on plants might not be as relevant here as the studies on algae. I know of a study on plants grown in the dark versus light, but I think that comes back to the NADPH, photosynthesis versus other pathways (OPP). To the best of my knowledge not published yet, but check.

For the C16:1 FAme it might indeed make sense to see if you can correlate changes in hydrogen isotopic composition to community composition changes since this does not show a linear response with time. For the others I think it would not be so useful.

Be careful with iso- and anteiso-C15:0 and C17:0 as indicators for all heterotrophic organisms and if they are only present in relatively low amounts in these organisms, heterotrophs could still contribute significantly to the other fatty acids.

Please go ahead and make the changes in response to the reviewers as you indicated you will and take my comments into account as well. I am looking forward to the next version of your manuscript.

Best regards,

Marcel

Dear Dr. Nemiah Ladd,

I agree with the reviewer that your manuscript has improved significantly and I would like to thank both reviewers for their thorough review and you for your detailed response and changes to the manuscript. I have a few small remarks for you to consider. I think I would prefer “..studying these effects under laboratory conditions…”(line 16/17). In line 20 of the abstract I’m not entirely clear what “these” refers to in “yet these remain poorly constraint”. Perhaps this sentence needs some work. At the end of the of the abstract to mention differences between the two lakes in brassicasterol and less so for phytol and you link this to nutrients. Besides (different) species producing these compounds, did you consider possible differences in IPP production pathways? On page 3, line 6 (axenic). On page 9 line 38, perhaps (a decrease in alpha lipid-water). As for heterotrophy during winter, page 11, heterotrophs typically follow their food, the primary producers, and also increase in numbers during spring and summer. The difference between summer and winter would be less an effect of heterotrophic contribution to the FA pool but more of a lack of autotrophic contribution during winter. For page 12, light intensity, temperature and growth of especially phototrophic organisms and, as a result of that, also heterotrophic organisms, are of course all related with each other. The two variable in line 3 of page 13, are these temperature and alpha?
I think if you address especially the typographical issues, rewrite a few sentences for clarity as indicated above and make sure the formatting of the tables is ok I think your manuscript can be accepted for publication in Biogeosciences.

Best regards,

Marcel