This work investigates the impact of different aerosolization techniques on E.coli within an atmospheric simulation chamber. The topic is surely interesting since it is relevant to many experimental protocols. Also, the analysis is quite thorough and bacteria viability is analyzed in multiple ways to better assess the impact of the aerosolization techniques. The reviewer suggests publication of the paper with only some minor modification as expressed below:

P. 3, Line 85: I suggest reporting also the aerodynamic diameter of E. coli (which should be smaller than 2 um) to make clearer the following choice of 0.5 um as a cut-off for the fragmentation range. 

P.5-6: Were the aerosol generators cleaned between replicates to avoid a carry-over effect in bacterial cells?

P.5-6: While the droplet diameter of the FMAG is reported (0.8-12 um) the one from the SLAG is not, is there an approximate size of the droplets and is it compatible with E.coli size?

P. 9: The WIBS peak is centered around 0.8 um vs. the OPS peak at 0.5 um. Couldn't this be simply an effect on how the different samples define the particles' binning? 

P. 9: Beside peak shifting WIBS seems to exhibit a higher (i.e.: non-overlapping error bars) maximum normalized concentration compared with OPS. Is this related to total particles counted by WIBS or to fluorescent-only particles? If so is that explainable by the dimensional shift or what alse could explain a higher number of fluorescent particles vs. total ones?

P. 10. Figure 3 is missing x-axis description (Midpoint bin (um)).

P. 14, L. 297-301. While the presented study is surely valuable and a thorough characterization, it is far from being the first step in this field. For example Thomas et al. (2011) already provided data on survival and site of damage of E. coli nebulized with different techniques. Rather, I think that the major strength of this work is the completeness of the analysis which goes way beyond simple viability (in a culturable sense). I suggest a rephrasing of this sentence.

Finally, this is not a comment on the quality of the paper itself, just an potential outlook for future work. Gram negative and gram positive bacteria differ in their structure, it would be interesting in the future to see if this also translates in differences when nebulized (in terms of viability, etc.).  

Cited literature:

Thomas RJ Webber D, Hopkins R, Frost A, Laws T, Jayasekera PN, Atkins T.2011.The Cell Membrane as a Major Site of Damage during Aerosolization of Escherichia coli . Appl Environ Microbiol. 77:.https://doi.org/10.1128/AEM.01116-10

This manuscript “Measurement Report: Effects on viability, culturability, and cells fragmentation of two bioaerosol generators during aerosolization of E. coli bacteria” provides valuable insights into bioaerosol sources for simulation chamber–based research. However, several major concerns remain. The authors are encouraged to address the following recommendations to strengthen the clarity, rigor, and interpretability of the manuscript.

Major points:

1) From the title and abstract, I understood that this study aims to provide a comparative investigation of two bioaerosol generators: the Sparging Liquid Aerosol Generator (SLAG) and the Flow Focusing Monodisperse Aerosol Generator (FMAG). However, the manuscript primarily evaluates the performance of the overall system, which includes aerosol generation, aerosol residence within the chamber, and subsequent bioaerosol sampling. As a result, the manuscript lacks a step-by-step evaluation that would help clarify which specific process(es) may be responsible for the observed effects on bacterial viability.

For example, lines 61–68 describe various stresses associated with the use of nebulizers; however, the nebulization step itself should be more explicitly isolated and evaluated. In addition, the remaining liquid within the nebulizer reservoirs after aerosolization should be examined more carefully to assess potential impacts of the nebulization process on bacterial viability.

2) The physiological status of the bacteria at each experimental step should also be considered more carefully. For instance, lines 290–291 mention … the bacteria probably tend to cluster, leading to droplets devoid of individual bacteria…. Such clustering could lead to prolonged bacterial viability, as cells located within aggregates may be shielded from environmental stresses. This protective effect could also result in higher CFU/mL values. Further clarification and discussion of this possibility would strengthen the interpretation of the results.

3) The nebulization and sampling periods (20–30 minutes) may themselves contribute to bacterial damage. Prolonged aerosol residence in the chamber, as well as high-velocity airflow into the BioSampler liquid accompanied by vortex motion, may impose additional mechanical stress on bacterial cells. These potential effects should be considered when interpreting the results, as part of an evaluation of the system as a whole.

Minor points:

Line 92–93: The manuscript states that 20 mL of bacterial suspension was centrifuged at 5000 rpm for 10 minutes. Please specify the applied relative centrifugal force (× g), as rpm alone is insufficient due to rotor-dependent variation. It would also be helpful to clarify whether the potential effects of this centrifugation step on bacterial viability or physiological status were considered.

Line 113–114: The particle size range is given as “0.55 ÷ 10 μm.” Please confirm whether the symbol “÷” is a typographical error and clarify the intended size range (e.g., 0.55–10 μm).

Line 147–148: Please specify the medium used to prepare the bacterial suspension for the nebulizers. A brief description of the bacterial solution, including CFU mL⁻¹ values before and after the experiments, would improve transparency and allow better assessment of potential losses during the experimental procedures.

Line 149: A nebulization duration of 20 minutes may be sufficiently long to affect bacterial viability. Please clarify whether bacterial viability within the nebulizer reservoir was assessed before and after nebulization. In particular, the SLAG system involves recirculation, which may impose additional stress on bacteria remaining in the reservoir. This potential effect should be considered when interpreting the results.

Line 156–157: The manuscript states that all experiments were performed at (22 ± 1) °C and (49 ± 1) % relative humidity. Please clarify whether these values refer to conditions inside the chamber. In addition, indicate how and where temperature and relative humidity were measured, and reflect this information in Figure 1 if appropriate.

Line 160: The BioSampler uses Milli-Q (MQ) water as the collection liquid and operates with a relatively high airflow rate into the liquid phase, conditions that may impose additional stress on bacterial cells. This potential influence should be discussed when interpreting bacterial viability and culturability results.

Line 186-189: … The results indicated that E. coli, resuspended in MQ, is not stressed during the short time required to run a chamber experiment (less than one hour). The potential loss of bacterial viability and culturability, as measured in the impinger liquid, can be attributed to the nebulization stress induced by nebulizers.…

 The physiological state of bacteria suspended in MQ water within the BioSampler is likely to differ substantially from the conditions implied by these statements and those represented in Figure 2. It remains unclear whether the bacterial concentrations measured in the BioSampler are directly comparable to those shown in Figure 2. Clarification on this point is necessary for accurate interpretation of the results. Moreover, the above statements (line 188-189) the suggest that bacterial stress and loss of viability may occur in both the nebulizer and the BioSampler. This overlap in stress sources reduces the ability to clearly attribute observed differences in bacterial viability solely to the nebulization process, thereby weakening the basis for comparing the performance and efficiency of the two nebulizers.

The introduction sets the context of the study effectively, in particular the lack of standardised approaches for bioaerosol research and the impact of aerosolisation and bioaerosol sampling methods on the viability of airborne microorganisms.  As with other areas of environmental microbiology, the issues of mechanical sampling stresses, also the question of viable, but non-culturable microorganisms and the biases these effects can bring to data collection, remain a challenge for aerobiologists.  Any information concerning bioaerosol generation and its influence on experimental findings is therefore helpful.

Lines 84-87 – General comment - As a test organism, the use of an E. coli strain was understandable, if challenging, given the relatively lower robustness of Gram-negative, non spore-forming bacteria compared to the Gram-positive species often used in aerosol test studies.  

Line 93 – as mentioned by at least one other reviewer, it would be helpful to have the centrifugation conditions expressed as xg to present a standardized value and to allow reproducibility, should the method be applied by others across different machines.

Lines 107-108 – the test chamber was small at 20l.  Even a class II biological safety cabinet would have been more specious and perhaps more representative of an ambient indoor atmosphere where particles can remain airborne at least for minutes prior to collection.  BSCs can also be cleaned very effectively, have HEPA filtered inlet air and have a reasonable internal volume, typically, of 0.8 to 1.0m³.  If not described elsewhere it would be useful if any perceived limitations of this very compact chamber choice were presented, compared with other obvious options such as a BSC, or just a larger steel vessel of the type used.  Maybe this could be added to the Conclusions section - see comment below?

Line 113 – I presume that the aerosol range should read, “…in the range 0.55 -10μm”, rather than “….in the range 0.55 ÷ 10μm.”

Line 115 and elsewhere – It would be useful to have the rationale for the choice of sterile water (for E. coli suspension and bioaerosol collection).  I can see that this might eliminate any risk of crystal formation, which might interfere with some of the applied assays, but the use of freshly prepared, sterile isotonic buffer (such as phosphate buffered saline) would perhaps have conferred increased protection for the aerosolised and sampled particles, and is perhaps the cell suspension medium that many others would have preferred.  Having read on, I do note that there is further comment on this between lined 169 and 189 and the journal editor may feel that this explanation is sufficient.  Figure 2 does go some way to indicating that viability was retained within the same order of magnitude (recoverable CFU/ml), throughout the short course of the experimentation.

Line 116 – The sampling pump flow rate is given as 12.5 l/min.  Given the limited test chamber volume did this have any implications for the testing?  For sample, very limited exposure of the bioaerosols to the airborne state prior to sampler entrapment?  From the test setup diagram I assume that any negative pressure effects in the chamber were avoided by use of the balancing effect of the HEPA filtered air inlet, but please comment on this further on the set-up if you can.

Lines 144-145 – Is this assumption based on the manufacturer’s information, or a fact established by other independent evaluation?  It would be useful to have that qualified in the text, or to modify the sentence in a way such as, “This modest shear stress typically allegedly allows biological cells to maintain viability, even following dispersion into uniform particles.”

Figure 3 required additional labelling on the x-axis - at least one other reviewer has commented on this.

Lines 297-301 - Conclusions section – are there any lessons learnt in terms of any experimental weaknesses identified?  The MQ water issue is well outlined, but there are few comments on experimental weaknesses or biases otherwise.  Also, the starting volume for each nebuliser is quite different – do the authors have any comments on the implications of this for wider aerosolisation testing and the practical choices to be made?  The paper of Gatta et al., 2025 is referred to several times throughout this manuscript; but are there any comparisons or reflections to be drawn between this earlier paper and the new manuscript.  As this earlier published work is clearly important to the current paper, it would be useful to get that perspective.