We thank the referee for their suggestions and their perspective. We would like to first address the concern about the presence of anaerobic respiration and its implications on our findings.

We agree that if meaningful amounts of anaerobic respiration occurred, that could drive RQ values greater than 1.0. However, there are several factors that lead us to conclude anaerobic respiration is  not the primary factor driving the elevated RQ values we measured, and these are listed as follows:

1) Soil headspace gas measurements were measured throughout the incubation, and even at peak respiration oxygen concentrations remained above 19% (these measurements can be found in the zenodo data repository).

2) The soil existed as only a thin layer within our bottles (no more than 1cm thick) and was comprised of particles <2mm in size. Given the strong inhibitory nature of oxygen on anaerobic respiration, we believe this would support abundant aerobic respiration and limit any anaerobic respiration. Further, the effect of sampling headspace and returning CO2-Free Air should effectively drive atmospheric circulation in the bottle.

3) Anaerobic respiration tends to occur more slowly than aerobic respiration, which further limits its possible impacts on our measurements.

4) The close agreement in the carbon budget presented in figure 5 suggests that aerobic respiration was driving the elevated RQ values we observed. Otherwise, during peak respiration ~50 % of the CO2 produced would have been produced through anaerobic respiration, meaning both anaerobic and aerobic respiration would be matched and this is not likely possible given incubation conditions.

 5) To account for RQ values around 1.5 like we observe, anaerobic respiration rates would need to be significant during peak respiration, when oxygen concentrations were still greater than 19%.

We acknowledge that we cannot entirely rule out the presence of any anaerobic respiration from having occurred; however, the data and the controlled incubation conditions imply that it was not significant in these incubations.

To address the second larger concern, the soil pre-treatment as described in line 136, was intentional to place the soil microbes in a stressed state with lower amounts of standing biomass so that when re-wet and amended with glucose, this would stimulate the production of new biomass in effort to measure its effects on RQ. This stimulation of biomass growth would require that bulk soil CUE would be greater than 0. CUE at peak respiration of around 0.4 is generally in line with other works which commonly range from 0.4-0.6. Many of these works, amended glucose to a relatively undisturbed soil in comparison to our soil pre-treatment which makes comparing them precarious. Though it is documented that with changes in environmental conditions, such as available substrate and soil moisture, changes in CUE do occur.

Changes in the text to address comments:

Line 36 - removed the word “around”.

Line 82 - sentence has been reworked to address ARQ first, then discuss the other non-metabolic processes, and added sentences addressing anaerobic respiration.

Line 94 -  “High temporal resolution” removed.

Lines 168 - 171 - Added statement regarding experimental design with intent to prevent and/or minimize anaerobic respiration from impacting our incubation results.

Lines 273-275 -  Follow up sentence, stating why this relationship can no longer be applied when RQ drops below 1.0, and what is likely occurring when this happens.

Lines 287-289- Added sentence discussing expectations for temporally dynamic CUE.

Lines 291-293- Added sentence discussing alternative explanations for RQ values and CUE as RQ values transition from >1.0 to <1.0.

Lines 311-313 - have been re-written more concisely for clarity.

Lines 319-320 - extended the sentence to mention that close agreement in Figure 5 suggests that anaerobic respiration was not likely an important process in these incubations.

Changes have been made to the document and will be updated with the next submission.

We thank the referee for their thoughtful comments and suggestions.

We believe that the applications of this method and relevant timescale of application are intimately linked questions. The exciting aspect of this approach is the relatively high resolution of these CUE estimates, which could be used to bolster our understanding of fast responses within the microbial community to environmental changes without the time intensive laboratory work associated with other methods for measurement of CUE. The other exciting aspect of this approach is that the metabolism in question can be tailored to answer different types of questions by using the same type of reaction-derivation, this can be applied to many other specific substrates. The second key aspect of this work is that we validate a theoretical link between CUE and measured RQ values. This knowledge will help provide context for field studies of RQ as well, now with the knowledge that transient shifts may be a result of microbial processes separate from changing substrates.

We believe this method will enable the observation of CUE at resolutions that would be considered logistically challenging with other methods, while the incubations we carry out are done with relative ease after the construction of the apparatus has taken place. We believe the types of scientific questions with which this method would provide strong advantage is the centered on this temporal aspect, for example studying the effects of dynamic environmental conditions on microbial metabolisms and carbon use efficiencies. Further, these incubations could be carried out for extended periods of time (several weeks to months) and provide relatively high resolution on not only CUE when substrate is well constrained, but for example study other processes which are predicted/known to impact measurements of RQ.  With minor modifications, this setup could be used in application to measure soil microbial respiratory behavior as oxygen is used up and conditions begin shifting to anaerobic, in effort to better understand the sensitivity and contribution of anaerobic respiration to bulk soil respiration.

Our thoughts are a single CUE that would best represent this period of glucose driven respiration would be a respiration weighted average of CUE over the period when RQ is ≥ 1. We believe this method is the most appropriate representation of CUE of the soil microbes specific to the period of time when glucose is the primary substrate.

We acknowledge that only considering C, H, O, and N is indeed a simplification of the real ecosystem and we hope that this work serves as a stepping stone and proof of concept. However, we are assured from Figure 5, that this fairly succinct simplification does effectively capture the process of biosynthesis in a manner which is approachable and compares very favorably with a carbon budget. However, further work could be done to provide an even more descriptive reaction to derive a more complete stoichiometric representation of soil microbes. However, it is also important to remember that other work has shown that microbial stoichiometry can vary with environmental conditions, so a more precise stoichiometry may be more applicable in certain use cases, but not all.  Additional elements that could be included in this are phosphorus or sulfur, however their stoichiometric contributions are quite small in comparison to C, H, O, and N.

Comments Addressed:

Multiple lines : fixed in text citations.

Line 110, 298: reformatted Equations.

Lines 113 - 139:  Moved appendix reaction derivation and added more derivation steps for readers to follow.

Lines 167 - 169 : Added sentence justifying our choice to add glucose as solid fine powder.

Lines 257, 258, 267-269: Added in figure numbers.

Line 329 : Added in reason for showing one replicate and method of choosing replicate, and why.

Lines 428 -  439: Added statements about how we think a prescribed CUE for the soils should be calculated and discussed the limitation of only using CHON elements to represent the system, as well as  which amendment sizes are recommended for further experiments and future applications of this research.

Changes have been made to the working document and will be included in the next upload.