We would like to thank Reviewer 1 for the insightful comments on our manuscript, “bg-2022-90”. We are glad that the reviewer finds our study on dark CO₂ fixation and its temperature sensitivity, to be interesting and that suggestions are given to improve the manuscript. 

To meet the reviewer’s concerns, we have addressed the question on why the temperature sensitivity of dark CO₂ fixation differs for rates reported per gram of soil and per gram of microbial biomass carbon and explained the rationale behind the quantification of the carbon allocation. We would also clarify working of all confusing text and remove Figure 7. Our point-by-point response to each of the reviewer’s comments is given below (see italicized bold text).

Reviewer 1: Comments to Author:

This study explores the temperature sensitivity of microbial non-phototrophic CO2 fixation in temperate forest soils. The manuscript is interesting but some aspects are not clear and require improvement. Particularly, the authors should explain why the temperature sensitivity of CO2 fixation differs depending on whether it is reported per soil mass or per microbial biomass C (see below).

Main comments

1. Figure 2 Why does Q₁₀ for CO₂ fixation differ between the rates per MBC and soil? I assume this is due to differences in the MBC in the two soil subsamples that have been exposed to different temperatures. It is rather surprising that the MBC concentrations differ so strongly, and it would be good to see the values (in a table).
   
   The difference in Q₁₀ based on rates per MBC and soil dry weight is not caused by differences in the MBC content as the MBC content were similar between the beech and spruce soils with hardly any changes with temperature (this is given in Table S2, supplementary information). Instead, it is caused by the difference of the rates being assessed. For calculating CO₂ fixation rates per gram dry soil, we measured the excess ¹³C in the soils and for rates per gram microbial biomass, we measured excess ¹³C extracted in MBC (lines 183– 195). Hence, we are looking at two different uptake rates: the rate of incorporation in total soil carbon (living and dead microbial biomass plus soil organic matter), and the rate of incorporation of the label into microbial biomass (lysable cells). The Q₁₀ values for both rates would be the same, if, for example, all ¹³C in the soils was still in the living biomass at 4 and 14 °C. 
   
   However, for the beech soil, we found 23% more ¹³C label incorporated into the MBC pool at 14 °C than at 4°C. In contrast, we found a 70 - 90% increase in ¹³C label incorporation at the higher temperature in the SOC pool. This led to larger calculated temperature dependence of fixation rates expressed per gram of soil and hence, higher Q₁₀ values, compared to those calculated for rates expressed per gram MBC. This information would be clarified in the revised manuscript.
2. Section 3.3 The rationale behind the quantification of the “C allocation” is not clear. Given that the incubation lasted only a few days, it is unrealistic that a lot of the microbial biomass C already turned into microbial necromass during the incubation. Thus, what the authors report here is probably rather the result of differences in the efficiency of the chloroform fumigation.
   
   We disagree with the reviewer that significant amounts of microbial biomass carbon cannot be transferred to SOM within the 21-day time frame of our experiment based on the different rates we measured. Our results show differences in the proportion of fixed ¹³C in the SOC and MBC pools for the beech and spruce soils (see reply to comment 1). Since CO₂ fixation is a microbial process, we assume that the excess ¹³C label measured in the soils after 21 days originates from CO₂ fixed by microbial biomass which has been transferred as microbial residues into the soil. Other studies found that microbial biomass can turn over quite rapidly as fast as 18 – 33 days in soils (Cheng, 2009; Spohn et al., 2016) and the transfer of microbial residues (both as turnover of necromass and formation of extracellular products from living biomass) into SOM can occur in as little as hours (Geyer et al., 2020). Since we don’t just expect microbial turnover via necromass production but also transfer of extracellular metabolites from living biomass, we would introduce the broader term, “microbial residues” in the revised manuscript which is by definition, any non-living organic material of microbial origin including necromass and extracellular metabolites (Geyer at al., 2020). 
   
   The reviewer is correct that differences in CFE efficiency might affect the calculated carbon allocated to MBC due to possible effects on MBC measurements. However, previous studies using a K_EC of 0.45 to account for the CFE extraction efficiency (Wu et al., 1990; Joergensen et al., 2011) as used in our study (lines 138 - 142), show that this factor does not strongly vary between soils irrespective of differences in soil properties (Martens, 1995; Joergensen et al., 2011) and would not be incubation-temperature dependent. Hence, we argue that the difference in turnover described now as residue formation between beech and spruce soils and also with temperature are caused by factors differentially affecting either the lifespan of microbial cells or the formation of microbial residues and this would be clarified in the discussion of the revised manuscript. Additionally, the relationships of rates to MBC previously found in other soils (Akinyede et al., 2020; 2022a) suggest that the CFE efficiency does not differ dramatically between the two soils. However, we cannot exclude possible effects resulting from differences in CFE extraction efficiency on our results. We will thus add a sentence in the revised manuscript to the effect that: While previous studies do not show that the CFE extraction efficiency factor of 0.45 varies strong between soils or temperatures of incubation, the assumption that this is constant between depths may affect our results, especially in comparisons of rates between different soil depths, or between rates expressed per MBC or gram soil.
   
   
3. Lines 459-461 I agree with this sentence. In addition, the authors should also mention that changes in primary production and root exudation might completely change the response of the studied processes to changes in soil temperature, which adds further to the uncertainty to the predictions. Given these uncertainties, I strongly recommend to remove Fig. 7 from the manuscript.
   
    We agree and we would include this statement in the revised manuscript and remove Figure 7.
   
   
4. Section 2.4 For how long were the soils explored to the ¹³CO2?
   
   In this study, all soils were exposed to the ¹³C-labelled CO₂ for a period of 21 days. we would modify the manuscript to clarify this.
   
   
5. L. 25-27 Based on the determined parameters (respiration and CO₂ fixation) no conclusion about microbial biomass turnover can be drawn.
   
   In reference to our reply to comments 1 and 2, we still wish to speculate about the microbial biomass turnover which we now describe as a part of microbial residue formation. But following the suggestion from reviewer 2 comment 1, this speculation would be limited to the discussion section of the revised manuscript.
   
   
6. L. 42 Remove “which also affects CO₂ emissions from other soils”
   
   This would be modified as suggested.
   
   
7. L. 52 Do you mean SOC concentration or quality?
   
   We meant both SOC content and quality. This would be modified in the revised manuscript.
   
   
8. L. 71 replace second “by” by “until”
   
   This would be modified as suggested.
   
   
9. L. 77-80 This statement cannot be drawn from the cited studies since they measured both processes at only one temperature
   
   Thank you for this comment. Our assumption is not only based on the findings from past studies showing that dark CO₂ fixation rates correlate linearly with net soil respiration (Miltner et al., 2005; Santruckova et al., 2018). We also considered that both soil respiration and CO₂ fixation rates increase with temperature as stated in lines 58 – 62 and lines 66 – 68. this section would be modified accordingly in the revised manuscript.
   
   
10. L. 83 are there other forest types in the temperate zone besides coniferous and deciduous forests?
    
    For simplicity, we would rephrase the sentence in the revised manuscript to reflect deciduous and coniferous forests as the two temperate forest types based on vegetation as reported in past studies.
    
    
11. Table 1 Please indicate the depths of the soil horizons
    
    This table would be modified as suggested.
    
    
12. L. 441/442 Remove “derived”
    
    This would be modified as suggested.
    
    
13. L. 462-464 These two sentences are not clear, at all.
    
    We apologise for the confusion; these sentences would be clarified in the revised manuscript.
    
    
14. L. 492-494 There seems to be some confusion here, and the process of microbial biomass turnover and microbial necromass turnover get mixed up. I think what the authors actually refer to is the rate at what C turns over in the living soil microbial biomass. It would be good to separate these two process more cautiously in the text.
    
    Thank you for this comment. We refer to the transfer or turnover of carbon from the living microbial biomass into the soil which we now describe more acurately as microbial residues (see reply to comment 2). Hence, we would remove the wording on necromass stability in the revised manuscript.
    
    

References of all articles cited in our response to Reviewer 1:

1. Akinyede, R., Taubert, M., Schrumpf, M., Trumbore, S., Küsel, K.: Rates of dark CO₂ fixation are driven by microbial biomass in a temperate forest soil, Soil Biology & Biochemistry, 150, 107950, doi:10.1016/j.soilbio.2020.107950, 2020.

2. Akinyede, R., Taubert, M., Schrumpf, M., Trumbore, S., Küsel, K.: Dark CO₂ fixation in temperate beech and pine forest soils, Soil Biology & Biochemistry, 165, 108526, doi:10.1016/j.soilbio.2021.108526, 2022a.

3. Cheng, W.: Rhizosphere priming effect: Its functional relationships with microbial turnover, evapotranspiration, and C-N budgets. Soil Biology and Biochemistry 41, 1795–1801. doi:10.1016/j.soilbio.2008.04.018, 2009.

4. Geyer, K., Schnecker, J., Grandy, A.S., Richter, A., Frey, S.: Assessing microbial residues in soil as a potential carbon sink and moderator of carbon use efficiency. Biogeochemistry 151, 237–249. doi:10.1007/s10533-020-00720-4, 2010.

5. Joergensen, R.G., Wu, J., Brookes, P.C.: Measuring soil microbial biomass using an automated procedure, Soil Biology and Biochemistry. doi:10.1016/j.soilbio.2010.09.024, 2011.

6. Martens, R.: Current methods for measuring microbial biomass C in soil: Potentials and limitations. Biology and Fertility of Soils 19, 87–99. doi:10.1007/BF00336142, 1995.

7. Miltner, A., Kopinke, F.D., Kindler, R., Selesi, D., Hartmann, A., Kästner, M.: Non-phototrophic CO₂ fixation by soil microorganisms, Plant and Soil, 269, 193–203, doi:10.1007/s11104-004-0483-1, 2005.

8. Šantrůčková, H., Kotas, P., Bárta, J., Urich, T., Čapek, P., Palmtag, J., Eloy Alves, R.J., Biasi, C., Diáková, K., Gentsch, N., Gittel, A., Guggenberger, G., Hugelius, G., Lashchinsky, N., Martikainen, P.J., Mikutta, R., Schleper, C., Schnecker, J., Schwab, C., Shibistova, O., Wild, B., Richter, A.: Significance of dark CO₂ fixation in arctic soils, Soil Biology & Biochemistry, 119, 11–21, doi:10.1016/j.soilbio.2017.12.021, 2018.

9. Spohn, M., Klaus, K., Wanek, W., Richter, A.: Microbial carbon use efficiency and biomass turnover times depending on soil depth - Implications for carbon cycling. Soil Biology and Biochemistry 96, 74–81. doi:10.1016/j.soilbio.2016.01.016, 2016.

10. Wu, J., Joergensen, R.G., Pommerening, B., Chaussod, R., Brookes, P.C.: Short Communication Measurement of Soil Microbial Biomassc Automated Procedure. Soil Biology and Biochemistry 22, 1167–1169, 1990.

We appreciate Reviewer 2 for the helpful comments on our manuscript, “bg-2022-90”. We are glad that the reviewer finds our study to be interesting with useful suggestions provided to further improve the manuscript. 

To meet the reviewer’s concerns, we would include more information in the revised manuscript, on the role of microbial communities in dark CO₂ fixation in the introduction, elaborate on the gene abundance results in the discussion and tone down information on the effect of clay content on microbial biomass turnover. We would, in addition, clarify all confusing wordings and texts on the sampling design and statistical analysis done. We have provided a point-by-point response to each of the reviewer’s comments below (see italicized bold text).

Reviewer 2: Comments to Author:

The authors compared the temperature sensitivity of dark CO₂ fixation and respiration in temperate forest soils of Germany. The fixed ¹³C was traced into microbial biomass and SOC, allowing the authors to comment on potential microbial biomass turnover rates under the contrasting temperature treatments. The study is interesting and the design is overall simple, but effective; there is limited information on the potential changes in dark CO₂ fixation under climate change. However, several aspects of the analysis and the discussion could be further clarified. Some of the results pertaining to the microbial community response and gene abundance were not adequately addressed in the discussion.

1. Line 26: This speculation around the role of clay content may not be appropriate for the abstract. Since the role of texture was not directly studied it is best not to highlight this as a possible mechanism in the abstract. Many other aspects of the systems may be able to explain differences in microbial biomass turnover. Similar comment for the last sentence of the abstract – “…variations in site-specific parameters might affect microbial biomass…”
   
   We agree and would limit our speculation on the role of clay content in microbial biomass turnover which we now describe as microbial residue formation (see reviewer 1 comment 2), to the discussion and remove this information from the abstract in the revised manuscript. 
2. Line 41-42: Add “through” so that it reads “through so-called dark CO₂ fixation…”. Also, what is meant by “which also affects CO₂ emissions from other soils”? This wording is unclear.
   
   Thank you. The word “through” would be added to the sentence as suggested. For clarity and in line with our reply to reviewer 1 comment 6, we would remove the phrase, “which also affects CO₂ emissions from other soils” from the applied sentence in the revised manuscript.
   
   
3. Line 66-67: What kind of ecosystems were included in this study by Nel and Cramer (2019)?
   
   This study was conducted for an afro-temperate forest and grassland ecosystems in Southern Africa, and this information would be included in the revised manuscript.
   
   
4. Line 71: I suggest changing “can warm” to “projected to warm”
   
   This would be modified as suggested.
   
   
5. Line 77-80: Please add a sentence or two to discuss the potential reasons why these processes would be expected to mirror each other.
   
   Thank you for this comment. In line with our reply to reviewer 1 comment 9, we would modify this section in the revised manuscript and change the wording “mirror” to clarify what we infer.
   
   
6. In general, the introduction could have more discussion of the microbial community’s role in dark fixation. 
   
   Following the reviewer’s advice, we would include more information on the role of microbial communities in dark CO₂ fixation to the introduction section of the revised manuscript.
   
   
7. Hypotheses appear to be implied in the writing, but could be explicitly outlined in this last paragraph of the introduction.
   
   Thank you for this comment. This would be modified as suggested.
   
   
8. Line 96-98: It is not clear what this is saying.
   
   We apologise for the confusion. Here we describe how the forest study site was established. We would modify the texts in this section to provide better clarity.
   
   
9. The beech and spruce plots were not replicated, correct? I am not sure it is possible to comment on statistical differences between spruce and beech plots without further replication of the forest types.
   
   We did not replicate each forest plot. However, during sampling, we took six replicate soil cores each from the beech and spruce plot (line 118). We later refer to these plots as “soil” and not “plot” in the manuscript and this number of soil core replicates are sufficient to compare these soils. For this reason, we would tone down our wording (in the discussion section of the revised manuscript) on what our results might mean for other beech and spruce dominated soils.
   
   
10. Line 126: What is a “biological replicate”?
    
    By using the term biological replicate, we refer to the replicates of the soil cores taken from each sample plot (either the beech or the spruce plot) and this is to account for variability within each of the sampled plots. However, for simplicity, we would only use term “replicate” in the revised manuscript.
    
    
11. Line 183: Should ¹²C/¹³C be ¹³C/¹²C?
    
    We apologise for the oversight. This would be corrected in the revised maniscript.
    
    
12. Line 279: What is the covariate in the ANCOVA?
    
    Thank you for this comment. When comparing some of the measured parameters between beech and spruce soil with ANCOVA, we included data from the whole soil profile for the beech and spruce soil. As a result, soil depth would also account for variability in these parameters. To account for this, soil depth was used as a covariate in the analysis. This would be clarified in the revised manuscript.
    
    
13. Line 504-506: The turnover may be slower in the clay-rich soil, but there is a greater availability of mineral surfaces that could potentially interact with C.
    
    It was not entirely clear to us what the reviewer’s point is here. The spruce soil with higher clay content had slower biomass turnover or more accurately, lower residue formation compared to the beech soil. The original discussion focused mostly on the potential interactions with clay that could slow the transfer of residues from microbial biomass to SOM in the higher clay spruce soil, including the idea that more mineral surface area could also contribute to this. We agree that clay can act in additional ways to explain the reduced transfer of label from microbial residues. For example, association with clay surfaces can lower residue formation in SOC if the production of microbial residues would be diluted by a larger overall SOC inventory created by the higher clay content. Thus, the amount of label transferred would be more diluted resulting in a smaller proportion of the total SOC. However, it is always difficult to say the exact mechanisms for the patterns we observed especially since many parameters were not measured or tested experimentally. Nonetheless, we would add a respective sentence in the revised version of the manuscript. 
    
    
14. In general, the authors should elaborate on the gene abundance results. There appears to be no comment on these results in the discussion.
    
    Following the reviewer’s advice, we would provide more information about the gene abundance to discussion section of the revised manuscript.