Peer review of: Microbial diversity drives carbon use efficiency in a model soil (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Domeignoz-Horta et al. present a work on the effects of biotic- and abiotic factors on carbon use 
efficiency (CUE). They generate a variety of novel soil microbial communities with a model soil and 
compare microbial CUE under different conditions of warming and drought. They found abiotic 
factors indirectly influence CUE through changes in microbial community diversity and 
composition, in particularly for soil bacterial community structure. Their results also indicate 
diversity-CUE relationships are context dependency, which can be modulated by soil moisture. As 
to my knowledge, this is the first empirical work using lab incubation to explore diversity-CUE 
relationships by considering several scenarios of climate change. This work is very interesting and 
would have great impact on the field of soil microbial ecology and climate change. However, there 
are some issues that need to address before publication. 
 
Major issues: 
1. Statistical analyses: 
(1) potential outliers of bacterial phylogenetic diversity under 30% WHC (values > 9; Figure 2) 
could influence of the bivariate association between bacterial phylogenetic diversity and CUE. It’s 
unclear whether the outliers have been considered in the processes of data analyses. 
(2) non-linear relationships are shown in Figure 2. Again, it’s unclear how the non-linear 
relationships are addresses in the structural equation model. 
(3) hypotheses presented in Figure 4 are too general. For example, what are the expected effects 
of community structure on CUE? The expected effects also apply for the other paths and need to 
justify but are not explicitly addressed in this version of manuscript. The processes for the model 
selection are also not clear. I cannot tell why the direct paths from moisture and temperature to 
CUE are not included. 
 
2. Result interpretation 
(1) Lines 200-201: This is a good point that nutrient availability and water content jointly influence 
microbial growth. But it seems like an uncompleted point. Why should I expected nutrient 
availability should contribute to microbial growth regarding to the incubation soil system? Please 
provide more information to explain this point. 
(2) Lines 221-223: Given enzyme activity measured in this study is potential activity (a measure is 
usually confounding with enzyme activity and enzyme production, the enzyme assays used cannot 
tease one from another), I don’t think enzyme production is the most likely reason to explain the 
negative effect of enzyme activity on CUE because this study does not exactly measure enzyme 
production. 
(3) Line 206: In this section, I am looking for the explanation for the negative effect of bacterial 
community structure on CUE, but I could not find it. This concern is consistent with the issue with 
the hypotheses of the SEM. I suggest the authors carefully present their hypotheses and provide 
reasonable explanation on the corresponding findings if possible. 
 
3. Implications: 
Lines 1-2: Microbial diversity driving CUE is based on the results of lab incubation. I am wondering 
how general I can expect for this work what are the implications in real world. These issues might 
be worth to discuss in the main text of the manuscript. In addition, microbial diversity is created 
by different experimental manipulations (D0, D1, D2, Bonly, SF). CUE could be also influenced by 
the experimental manipulation. That is, there is one alternative that has not consider in this 
manuscript, i.e., microbial diversity could co-vary with CUE, rather than microbial diversity drives 
CUE. Finally, the experimental design could also allow to quantify the unique contributions of those 
abiotic- and biotic factors on CUE, as well as the fungal and bacterial contributions on CUE, it 
might be good to explicitly present these potential mechanisms. 
 
Minor issues: 

Lines 18-20: This result need to be re-considered after excluding the outliers in low moisture. 
Line 23: Why would I expect climate change would have negative effect on the provision of 
ecosystem functions? For instance, climate warming might increase forage production. 
Line 39: It might be good to justify why should I care about temperature and water availability 
given there are several abiotic factors could influence CUE simultaneously. Is that because the two 
factors can be easily controlled in the lab? The rationale is unclear here. 
Line 82: I know there are 200 samples in total considered. But it’s hard to tell how many samples 
they used for the specific analyses. For example, Line 284, the SF treatment at 15 oC and 30% 
WHC is excluded. Will this influence the results or interpretation? Line 318, how many negative 
samples are excluded? It would be good if the authors provide the exact sample size for their 
statistical analyses. 
Lines 105-106: I don’t understand the median fungal richness was zero. That means negative 
fungal richness is also observed, right? 
Lines 114-115: this sentence does not seem like a good fit to the results section. I am wondering 
how I can understand the same density reached. 
Lines 125-127: What does this mean the level of diversity? It might does not make sense to 
compare this study with the other studies. 
Lines 150-152: Is this a common level of community diversity (<10 species) in natural 
ecosystems? Sounds like the review is based on biodiversity experiment. 
Line 258: typo in base media? with. 
Line 268: 120oC? 
Lines 273-274: I still do not follow the rationale of the experimental design. Why 30 or 60% WHC 
and 25 or 25oC? What do the experimental settings mean in real world? 
Line 280: added substrate no substrate? 
Line 299-301: I don’t understand this sentence. 
Line 313 and 326: Qiagen? 
Line 332: I don’t understand why NAG and BG are related to P cycling. 
Line 357: Not clear. What do you mean same DNA extracted for CUE estimates? 
Line 387: Comparative Fit Index? Are you sure you can obtain this index from piecewiseSEM? 
Line 466: Author names are missing. 
Line 478 and 504: Journal names are missing. Please check though the references. 
Figure 1: delete one between. 
Figure 2: What do (a), (b) and (c) really represent in this figure? 
Figure 3: grown at grown? 
 
I hope my comments and suggestions would be helpful for the revision of the manuscript. 
 
Xin Jing 
2020.01.14 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript by Domeignoz-Horta and co-workers investigates how microbial community 
metabolism (as measured by C-use efficiency (CUE) = ratio of growth over substrate consumption) 
changes in response to different abiotic conditions (temperature, soil moisture) and microbial 
community diversity or composition. This work is timely, as most other papers on this topic focus 
on abiotic conditions as drivers of microbial metabolism, and less on microbial community 
characteristics. I find the experimental design adequate, but the description of the approach is not 
always clear. I also have several relatively minor comments. 
 
- Methods: I would clarify in the Methods why a short-term experiment is conducted – it now reads 
as if the short-term experiment is just in addition to the long-term one. The motivation is there 
(around L307), but somehow hard to follow. Moreover, it is not clear why the dry D0 treatment 

was used for the short-term incubations. Also, see comments below regarding some regression 
analysis – data are clustered and a regression model seems to show strong bias at high diversity 
levels. 
- Interpretation of aggregate-CUE relations: aggregation is positively correlated to CUE in the dry 
treatment, but the explanation provided in L167 that growth was limited by resources in 
aggregates is not convincing. Were growth and respiration rates correlated to aggregation? Was 
the C or nutrient content of aggregates measured to support this explanation? 
- Figure 1 shows the soil samples with apparent microbial growth – this makes me wonder if the 
biomass and C fluxes measured in the long-term incubations are comparable to those that would 
be measured in natural soils. I realize this is a study based on idealized conditions, but it is 
relevant to discuss similarities and differences compared to the real world. 
 
Other comments 
L85: at this point in the manuscript, it is not clear how aggregation is connected to substrate 
supply –some explanations are needed 
L102: in the Methods (P13, L265), dilution is reported to be of four (not five) orders of magnitude 
L138: does “scaled” mean “increased”? 
L153: perhaps “is consistent with” instead of “corroborates to”? 
L157: this hypothesis is a bit far fetched and not testable here – especially the link to CUE – 
because diversity is manipulated but not complementarity. The hypothesis is plausible, but some 
more explanations of why CUE should increase when species are complementary are needed 
L179: typo “effect on CUE” 
L181: I do not see the connection between these sentences. The premise is that in the long-term 
it is difficult to separate abiotic vs. biotic drivers on CUE. As explained elsewhere in the 
manuscript, long-term and short-term experiments tell different parts of the story, but a short-
term experiment won’t help disentangling the long-term abiotic vs. biotic effects on CUE 
L187: why selecting the dry D0 treatment? 
L247: or the other way around? From previous statements and structural equation modelling, it 
seems that abiotic factors are acting indirectly on microbial metabolism, the effects being 
mediated by microbial community characteristics – so it is the abiotic effects that are masked by 
the biotic ones 
L248: this work shows that microbial diversity matters, but why “more than previously believed”? 
this seems a bit of an overstatement… 
L264: shouldn’t the inoculated amount be expressed as a volume of inoculum solution per gram of 
model soil? Or was ‘real’ soil mixed with the model soil? Please clarify 
L275: higher WHC should correspond to higher (less negative) water potential 
L305: I would add a short sentence to explain that in addition to the long-term incubations, you 
also want to perform short-term incubations to test abiotic effects on rates independent of 
community changes. It is stated later, but I found this transition not very clear 
L337: typo “establish” 
L334: typo “capillarity” 
L348: the sentence starting here is convoluted and hard to follow 
L466: reference missing author list 
Figure 1: please check dilution value – 5 or 4 orders of magnitude? 
Figure 2 (also some figures in SI): data points are clustered at low diversity levels, and variance 
does not seem homogeneous – how were these inhomogeneities in the data treated when 
conducting the regression analysis? Also, the CUE function in the bottom right panel seems to 
completely miss the high CUE values at high diversity, suggesting that the model chosen for the 
fitting is not appropriate 
Figure 3: I would suggest highlighting the bars in the box plots that correspond to unchanged 
abiotic conditions in the long- and short-term incubations. These are reference points for the other 
bars