Frequency of disturbance alters diversity, function, and underlying assembly mechanisms of complex bacterial communitiesEzequiel Santillan, Hari Seshan, Florentin Constancias, Daniela I. Drautz-Moses, Stefan Wuertz, May 4th 2018, bioRxiv[doi: https://doi.org/10.1101/313585] Understanding the effects of disturbance on ecosystem function and diversity has many potential applications in microbial ecology and human disease biology. In this paper, the authors tackled the long-standing question of how different disturbance frequencies affect bacterial community diversity and function. To do so, activated-sludge communities within laboratory-scale microcosms were exposed to toxic 3-chloroaniline (3-CA) at varying frequencies. Ecosystem function and community diversity were measured weekly by measuring biomass and organic carbon, ammonia, and toxin removal as proxies for ecosystem function and T-RFLP 16S rRNA gene fingerprinting and shotgun metagenomics were performed to examine variation in bacterial diversity and community composition. This work is an excellent example of integrating genomic and functional analysis, thereby providing a more thorough understanding of the effects of disturbance frequency on microbial community diversity and function. Interestingly, both genetic methods yielded similar results, suggesting that the less expensive gene fingerprinting method could be sufficient when sequencing resources are limited. We particularly commend the use of multiple alpha-diversity measurements and the inclusion of abundance-related indices, which are less method dependent and allow results to be compared between studies. Ultimately, the authors propose the "Intermediate Stochasticity Hypothesis,” which suggests that stochastic processes produce higher diversity assemblages at intermediate disturbance frequencies while deterministic processes produce lower diversity assemblages at low and high disturbance frequencies. Overall, this paper is a fascinating and substantial contribution to microbial ecology. There are, however, a few issues that we feel could be improved in future versions of the manuscript.   Major concerns:This comment is unique to the preprint. The manuscript references multiple figures available in the supplementary materials, but these materials were 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 materials to bioRxiv. 1. Figure 2 is an integral figure to the manuscript because it showcases the effects of 3-CA disturbance frequencies on community performance, namely organic carbon and toxin removal (plots A, C)  and nitrification products (plots B, D). In the Materials and Methods section (lines 353-356), the authors state that these parameters were measure weekly, which leads to the assumption that data is available for days 7, 15, 21, and 35, even though only the data from days 7 and 35 are included in the figure (is there T0 data?). The results section refers to supplementary figures S2 and S3 in addition to Figure 2, so these supplementals may portray the data of interest. However, since these data are so important to the overall conclusions, we believe it should be available in the main text. One way to accomplish this could be to have one plot per variable with time on the x-axis and different colors for each disturbance frequency. The number of plots could be reduced by not including Volatile Suspended Solids (VSS) results in the main text. In Figure 2A, the COD removal and 3-CA removal is not monotonously decreasing relative to the disturbance frequency (specifically, level 2 and 4). We figured that this was due to the number of days since the disturbance being different for each disturbance frequency at measurement time on day 7. We encourage the authors to mention and explain this in the text, as this was a puzzling feature of the results for us for some time. It also calls into question the appropriateness of the weekly measurements, especially given that some disturbance level will be highly correlated to this rhythm of measurement (level 1 disturbance will always happen on the same day of the measurements, while level 2 and others will drift). 2. Along with disturbance frequency, varied intensity and duration of disturbance and differing sampling frequencies (e.g- data collection every two days or  bi-weekly, larger spread of intermediate disturbance levels) might produce 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? We would be very interested in the authors opinions on these topics, perhaps in the discussion section.