This manuscript uses cultures of two Thaumarchaeotal strains to investigate growth temperature and phase on OH-GDGT distributions and OH-GDGT-based proxies. This is an important piece of work in the context of the emergence of OH-GDGTs as a GDGT-based temperature indicator, especially in cold water settings. The paper is clear and well written with nicely presented figures, and thoroughly reported results. I mostly only have a few very minor suggested comments/changes, but did feel like the manuscript was missing an ‘implications’ or ‘summary’ section to tie the results together and put them into a wider context. Could the authors comment on what they see as the key implications of interspecies variability in OH-GDGTS with changes in growth temperature for OH-GDGT-based temperature proxies? Specific comments below:

Line 23. Add ‘the’ before natural.

Line 32. Add a comma between ‘moieties’ and ‘and’.

Line 43. Add ‘the’ before Black Sea.

Line 141. Correct format for tex86oh.

Figure 3: Is there a reason purple shaded boxes haven’t been added to c) and g)? It would be good to see how these results compare to global core top data sets for RI isoGDGTs.

Line 347: Extent rather than extend.

Discussion: It feels like an implications section is missing here, that sums up the results and discussion and distills out the key points and implications of these findings for future OH-GDGT- based research. I recommend adding a paragraph to this effect before the conclusions.

This manuscript systematically investigates the response of hydroxylated GDGTs (OH-GDGTs) in ammonia-oxidizing archaea to temperature variations and different growth phases, revealing several significant findings. The manuscript is well-written, the data are presented clearly, and the conclusions are substantial and scientifically meaningful. However, I have two main concerns and a few suggestions for further research:

1. The study uses the Bligh-Dyer method for lipid extraction, and it is mentioned that this method may lead to partial dehydration of hydroxylated isoGDGTs, potentially underestimating the abundance of OH-isoGDGTs. This could partly explain the discrepancies observed between experimental data and environmental samples. I recommend that the authors discuss the potential impact of this extraction method on the results more thoroughly and consider whether alternative extraction methods might more accurately reflect the GDGT composition in pure cultures. I would recommend considering the use of cellular acid hydrolysis in future studies to investigate the response of core lipids systematically in the future. This approach may provide a more comprehensive understanding of how core lipids behave under different conditions and could help to address some of the limitations observed with the current extraction methods.

2.  Figure 1 shows that the N. piranensis D3C strain exhibits some differences at higher temperatures, particularly at 35°C, where the relative abundance of OH-isoGDGTs differs significantly between growth phases. However, the discussion of this aspect in the manuscript is somewhat brief. I suggest the authors provide a more detailed explanation of the mechanisms by which high temperatures might influence these differences, especially when compared with environmental observations.

3. Given that the study found inconsistencies between the behavior of OH-GDGTs in the experimental setting and in environmental samples, particularly regarding temperature's effect on cyclization but limited impact on OH%, future research could focus on the performance of different strains at lower temperatures (<15°C), especially those isolated from cold/polar regions. This could provide valuable insights into the physiological role of OH-GDGTs in archaeal membrane adaptation and further refine the proxies based on them.

I believe that this manuscript makes a significant contribution to our understanding of OH-GDGT proxies and is worthy of publication.