Does the title accurately reflect the results?
Experimental design: were the techniques appropriate for the hypothesis? Were suitable controls included? Were new techniques/mutants/cell lines etc. sufficiently characterised to allow the study to be replicated? Were robust statistical methods used?
Do the results support the authors’ conclusions and, if not, what additional experiments would you suggest?
Are there big flaws in the understanding of the current literature and interpretation of the literature that might undermine the interpretation of the current results?
Major concerns-
0) The manuscript references multiple figures available in the supplementary data which was not made available as part of the preprint. This hindered our ability to understand the fine points of the experiments. We encourage the authors to upload the supplementary data to bioRxiv.
1) The figure 2 is an important figure showcasing the effects of 3-CA disturbance frequencies on organic carbon (plots A, C), biomass (plot C) and nitrification products (plots B, D). We would suggest the authors to make the functional data for Day 14 (currently available in the supplementary Figure 2) in the main paper, which will make the observed correlations on Day 35 easier to understand.
Similarly, it would be interesting to have a PCoA plot of an intermediate day in Figure 1 for continuity of message and to support the authors' results of observed patterns on Day 35.
2) The paper was a very interesting read and we had a very lively discussion about what kind of measurements, we as readers would like to see in the paper. So, we would like to suggest some improvements in the experimental design-
- Addition of data points before and after 3-CA disturbance was introduced at each time point. This will help illustrate that the observed functional changes were indeed caused by the introduction of disturbance.
- Representation of time t=0 when the microcosm experiment commenced to check if the microbial diversity and functional variation caused by disturbances were different from the initial state of each community.
- The frequency of measurements implies replacement, we'd like to see a discussion about how replacement was achieved and what the impacts of replacement may have been. We'd also like to see some discussion on the implications of scaling up the microcosm size and varying initial conditions to reproduce and expand the experimental design for further work testing the new model.
- Along with weekly disturbance levels, varying intensities and duration of disturbance (e.g- data collection every two days or bi-weekly, more spread of intermediate levels) might have a different pattern of microbial community diversity and function. Questions we can ask are, would the system reach the observed IDH pattern at an early stage? Would the intermediate levels still follow the IDH model?
3) We would welcome the use of different statistical tests for data analysis and visual colours to represent different disturbance levels. We would like to suggest the addition of distinct data labels for each of the disturbance levels. For example in Figure 3, along with using identical colours in A, B and C plots, usage of identical data labels in C would help make the figure more interpretable.