This discussion paper uses carefully controlled experiments to demonstrate the temperature sensitivity of microbial carbon use efficiency and how the isotopic composition of respired CO2 can change with temperature and substrate use. The manuscript is novel because it bridges concepts that are of current importance in both terrestrial and aquatic biogeochemistry, with relevance to carbon cycling on scales up to the globe. Temperature dependence of C use efficiency is a major uncertainty in global models but is a critical determinant of rates of C storage or loss with climate change. The methods are novel because they combine standard chemostat techniques with newer continuous-flow stable isotope measurements; the diagram and detailed description of steady-state tests in the supplemental material will allow for replication or modification of these techniques.
Two experts agree that the manuscript offers substantial insights but that it requires revision before it’s ready for publication. The main concerns are related to uncertainties or potential errors associated with the measurements, which need to be addressed in the revised version. We encourage the authors to submit a thoroughly revised manuscript, as described in the detailed and thoughtful response to reviewers.
As suggested by Reviewer 2, Figure 5 should be modified to demonstrate how this controlled experiment has provided some insights into natural systems, and what unknowns remain to be constrained. Can you demonstrate the relative uncertainties in the unmeasured fluxes in the chemostat system? It’s not clear why you give the d13C value of the substrate but not the other pools or fluxes; maybe conceptualize the fractionation you observed somehow.

This paper provides insights into the temperature dependence of microbial carbon use efficiency and potential C isotopic fractionation associated with heterotrophic respiration in the context of unlimited substrate supply. The revisions have improved the manuscript by increasing the “reach” of the paper to both terrestrial and aquatic biogeochemists.
The authors are encouraged to add a short paragraph that could provide some guidance for others seeking to follow up this research (“next steps”). This section could include some suggestions regarding replication, the role of unlimited substrate concentrations in detecting isotopic fractionation, alternative substrates and microbes, applicability to real-life aquatic or soil systems, etc.
Specific comments:
Please check all figures for units: It seems likely that specific microbial growth and respiration rates are presented in mg C per g microbial biomass C, but please make this clear.
Line 329, I think the difference should be 7 permille (not 10).