In this work, Jabinski and colleagues quantify the water assimilation factors and heterotrophic incorporation of inorganic carbon into biomass, using fungal cultures. This “dual-SIP” approach is useful for calibrating environmental SIP approaches. I have no major comments on this work and recommend it for publication. I have detailed some minor comments below that should be addressed prior to publication.

Section 3.2.2 (and throughout): the water assimilation factor should be noted as a_w, not alpha_w, to avoid confusion with alpha fractionation factor notation.

Figure 3: the units of the x and y axes should be cleaned up. For these values I would recommend ppm.

Line 58: Please add the following references here:

Warren 2022 “D2O labelling reveals synthesis of small, water-soluble metabolites in soil.”

Caro et al. 2023 “Hydrogen stable isotope probing of lipids demonstrates ...”

Canarini et al. 2024 “Soil fungi remain active and invest in storage compounds during drought independent of future climate conditions”

Line 180: Make sure to subscript CO_2.

Line 287: An a_w of 1.2 is nonsensical, no? I was curious and looked at the figure I believe you are referencing, and the histogram bins only go to 1.0. It would be more accurate here to say something along the lines of “A_w can theoretically vary between 0 and 1, representing conditions where an organism acquires none or all of its lipid H from water. The consensus from previous studies is that microbial heterotrophs exhibit a_w values typically around 0.71 +/- 0.17.”

Fig. 5: It is unusual to have a figure included in the conclusion section. I would recommend switching the position of this figure to the main text. Furthermore, it is currently unclear what this figure adds to the manuscript. The color and shape scheme is very difficult to parse and should be cleaned up, or the figure should be removed.

Jabinski et al. cultured fungi from several species with 2H-labeled water and 13C-labeled DIC. The fungi took up very little inorganic carbon, indicating that they were primarily heterotrophs and ate the unlabeled organic carbon provided in the cultures. Apparent ²H/¹H fractionation factors varied among species and substrates, but I don’t think the alpha values reported for these were calculated correctly (although what the authors have done is consistent with some other publications, I don’t think it is appropriate when the product and the substrate are separated by multiple ²H-fractionating reactions, as explained below). The discussion was also a bit shallow in terms of integrating these new results to other publications that have investigated the relationship between metabolism and ²H/¹H fractionation. The emphasis is more on the utility of the dual-SIP approach, and it seems like there is a bit of a missed opportunity to discuss how these results can inform our understanding of fungal metabolism. I think the data are unique and I appreciate that the authors have put in a large amount of work to generate them. Very little is known about 2H/1H fractionation by fungi, and this has the potential to be a useful tool to understand fungal metabolism in soils. I think with some rewriting, this manuscript will be a helpful contribution to this field.

Best wishes,

Nemiah Ladd

Specific comments:

Line 27: this fractionation factor is actually much lower than the values typically seen for bacterial heterotrophs, and I think is due to the way you have calculated alpha (see comment below)

Lines 107, 117, 128, 139 "analytical error": specify if you mean accuracy (measured offset from known value) or "precision" (standard deviation or standard error of replicate standard measurements)

Line 166: would be good to specify both precision and accuracy for these measurements. Were samples measured more than once (in duplicate or triplicate)? If so, can you report average standard deviation of replicate sample measurements?

Section 3.2.2, Figure 3: I’ve seen several instances where ²H/¹H fractionation factors between lipids and water are calculated from the slope in this way, but I don’t think it works to calculate alpha from the slope if there is more than one reaction separating the product from the substrate, as is the case for fatty acids synthesized from water. If there was a single fractionating step, the alpha value calculated from the slope would correspond to the epsilon value calculated from the y-intercept. This doesn't seem to be the case for your data. See Sessions, A.L., Hayes, J.M., 2005. Calculation of hydrogen isotopic fractionations in biogeochemical systems. Geochim. Cosmochim. Acta 69, 593–597. It would be better to calculate alpha from each lipid-water pair and report average apparent fractionation factors per species. At a minimum, you should provide the full equations for each linear regression that is shown, including the y-intercept and not just the slope. There is a statement that the data set will be published, but it is not available for reviewers to see, so I’m not able to check this myself.

Section 4.1, Figure 4: shouldn't the CUE results be presented already in the results section, not in the discussion as they are here?

Line 282: see also a recent paper about H isotope fractionation by yeast by Ashley Maloney et al. (PNAS, 2024), which is relevant here since it is one of the few studies to look at H isotope fractionation in fungi

Figure 1, subsequent figures: It would be nice if all six species were indicated in the legend, rather than the three genera. It is a little confusing how half of the colors in the figure are not shown in the legend, and you have to read the caption to make sense of them. Then for the other figures that use the same color scheme, you always have to refer back to the figure 1 caption to figure out what they mean.

Technical corrections:

There are several places where articles (e.g., the) are missing or where they are used when they should not be. I’ve noted some examples of this below, but I suggest that someone read through the manuscript carefully and correct this throughout.

There are also many cases were subscripts or superscripts are missing (e.g., CO2, 13C)

Line 39: Add "the" before "atmosphere"

Line 53: Awkward phrasing, I suggest editing to "now allow microbial taxa to be linked to specific processes..."

Line 106: missing articles (need "a" before "standard" and "laboratory"

Line 108: Here, I think you don't need "the"

Line 151: Add "The" before "transfer"; ion source should not be capitalized

Lines 201-208: I would move the equations and the descriptions of them to the methods

Line 276: I think you mean to refer to figure 4 here?