General comments:

Using a series of bulk laboratory experiments, Liu et al. investigate the effects of acidity and light exposure on the survival and metabolism of two bacterial strains (isolated from an aerosol sample) in cloud water. In particular, they measure the culturable cell concentrations and ADP/ATP ratios to calculate the survival rates and energetic metabolism of cells, respectively, under various combinations of acidic and illuminated conditions. UPLC-MS was used to measure water-soluble and -insoluble biological material from cell lysis and IC was used to measure carboxylic acid concentrations and calculate biodegradation rates. Based on their measurements, Liu et al. concluded that biodegradation could be competitive with chemical oxidation under certain conditions. They summarized their results by stating that cloud water pH has an effect on the survival and metabolism of neutrophilic bacteria and that combinations of stressors can be more significant than expected from measurements conducted in isolation. While the experimental design and physical measurements seem generally sound, this work raises several questions regarding atmospheric relevance, which are specified below.

Specific Comments:

• Pg 4, ln 122. Please elaborate on how and where the aerosol samples, from which the bacterial strains were isolated, were collected. For example, is the altitude or temperature of the sampling location known?
• Please comment on whether the bacteria were metabolically active in the atmosphere or rather on the possibility that they might have been dormant. What implications does this have for the results?
• These experiments were conducted in 5 mL volumes based on Methods section 2.2 (pg 6, ln 176). Multiple points in the manuscript (e.g., pg 6, ln 179; pg 7, ln 196; pg 8, ln 226) mentioned aliquots of the sample being removed for analysis. What volume was removed, and would this in anyway bias the results? Were the test tubes stirred over the course of the experiments, and if not, could this impact the data?
• Is there evidence that bacteria were actively metabolizing in the acidity and light experiments if the decay of organics in the cloud water mimic could not be observed? Have any live/dead staining before and after light exposure been performed?
• Pg 8, lns 220-222. While citations are provided to support the claim that the concentration ratio between chemicals and cells rather than absolute values are important for degradation rates, the latter two both reference the first one (Vaïtilingom et al. 2010). In that case, it is for a different bacterium and for a single carbon source. Can their assumption be extended to these experiments given the discrepancies?
• What were the “simple” (pg 17, ln 410) calculations used to create Figure 5? Are they merely ratios of the degradation rates or were they based on a model? Either way, what are the assumptions used and the limitations of that approach? 
• Furthermore, although Fankhauser et al. 2019 is referenced (pg 16, ln 379; pg 21, ln 478), their main finding was not acknowledged. They concluded that metabolically active microorganisms in the atmosphere are physically separated from the majority of atmospheric water (and thus organics), and that bacterial metabolism should not significantly affect the total organic content. This seems to directly contradict the finding that bacterial metabolism can be competitive with chemical oxidation. How can this be reconciled?

Technical comments:

• Pg 7, ln 183: Please define ADP and ATP.

Comments on Liu et al. : « Effects of pH and light exposure on the survival of bacteria and their ability to biodegrade organic compounds in clouds: Implications for microbial activity in acidic cloud water”

Microbial activity in clouds have been shown to potentially influence cloud chemistry. This study investigates how pH in cloud water modulates the survival and metabolic activity of 2 bacteria (Enterobacter) species isolated from air samples, and their capacity to degrade selected organic compounds. The topic is still indeed untreated, although it could have important implications regarding atmospheric processes.

Model strains of atmospheric isolates were grown in the lab and subjected organic compounds in a solution resembling bulk cloud water, at different pH (4.2, 4.3 and 5.9) and in the presence or absence of light. The biodegradation rates of atmospherically relevant organic compounds (formate, acetate, oxalate, maleate, malonate, glutarate and MSA) were investigated under the different conditions of light and pH, and the loss rates compared with those due to chemical degradation involving radicals.  The results show that pH is an important factor that could impact biological survival and activity, and that synergetic effects exist between the 2 variables. The results also confirm pH and light have different impacts on survival and biodegradation rates depending on the microbial strain.

The manuscript is written in correct language, but there is a general lack of precision in the statements (MSA is not a carboxylic acid for instance), important references are missing or misinterpreted (such as Khaled et al. (2021), where the necessity to account for the multiphase and heterogeneous aspects of clouds is emphasized, or the (inexact) statement that biodiversity in clouds was only investigated so far through culture methods L60. In addition, in several places the interpretations could be expanded and contextualized.

I had difficulties to figure out what was the rationales behind some aspects of the study that not seem to bring additional information, and that are not discussed into context, such as ADP/ATP data and the list of organics released during cell lysis. The latter in particular appears totally disconnected from the rest of the study, barely out of subject, while it could be presented and discussed as a source of organics to cloud water, and/or biomarkers of cell lysis. Are these compounds indeed found in natural cloud water? Could these be used by the remaining active cells as substrates? Could these be used as biomarkers of damages to cells in such environments?  About ADP and ATP, could these be used as proxies to evaluate survival and biodegradation rates in natural situations? Does this variable provide additional information here compared to cultures regarding survival?

The genomes of the bacteria investigated were sequenced, and such data could provide useful information to interpret the data. Nevertheless, genomes have not been exploited at all in this study. Looking for relevant enzymes and functions in the genomes (pH homeostasis, internalization and use of carbon substrates, etc) and discussing them would definitely strengthen the paper regarding the biological aspects of cloud microbiology.

It is not clear at what time points the biodegradation rates were calculated from experiments. Were [cell]_experiment adjusted for accounting for the decrease of survival? In this regard, it is indicated (line 428) that “a constant bacteria concentration of 8 × 10⁷ cell L^-1 was assumed in our calculations”. First, it has to be clear that this number is the highest estimate of the actual active cell concentrations (i.e. 100% of active cells), as these correspond to total cell numbers in the references cited, and second: it appears odd to consider active cells constant in acidic clouds while at the same time presenting data showing that survival and activity are affected. This aspect would need at least a bit of clarification and discussion.

Besides survival, there are likely other aspects related with pH that could be considered/discussed: how it modifies the form of the organic compounds studied (pKa) and so their biological availability? Can this impact the solubilization and volatilization of the organic compounds in clouds, and so their biological use in cloud droplets? Additionally, different organic compounds were mixed together in the incubation medium. Was a prioritization observed, i.e. were some substrates preferentially used over others?  Your work could provide valuable information here.

Absence of statistics: Statistically significant differences are mentioned (L391 and elsewhere), but the tests used and the results are not specified; these should be.

About modeling, the approach used is quite similar as early work regarding the evaluation of the impacts of biological activity on cloud chemistry (e.g. (Vaïtilingom et al., 2011). This basically consisted in comparing the biodegradation rates to radical chemistry rates. However, such a simplistic approach totally omits the main specificity of cloud water, i.e. its distribution in droplets. This is my main concern regarding this work. Yet distribution in droplets has been shown to be a key factor in assessing the impacts of biological degradation in clouds (Khaled et al., 2021), particularly because not all droplet contain bacteria cells, and because water-air exchanges are critical in such systems. Therefore, the predicted relative contributions of bacteria and radicals to the loss of organic compounds in this work are ultimately valid only for the population of droplets containing bacteria cells, but not for the entire cloud. This should be clearly stated and if possible, this part of the work could be reevaluated.  

Specific comments:

L46: what are “microbiological-ecosystem interactions”? these are likely not precisely atmospheric processes.

L54: why “however”? there is no contradiction here.

L57: “The cell concentrations of metabolically active bacteria in clouds typically range from about 10² to 10⁵ cells mL^-1”: these are numbers for the concentration of total bacteria, not only those metabolically active.

L60-61: The authors should read the references that they cite, by far not only culturable bacteria have been investigated in clouds: (Amato et al., 2017, 2019) rely on molecular data (rRNA and rDNA), and reference could also be made to (Péguilhan et al., 2021).

L66: (Zhang et al., 2021) does not investigate microbiological-chemical interactions but physical impacts of bioaerosols.

L67: “Many bacteria species isolated from cloud water have the enzymes needed to biodegrade organic compounds such as carboxylic acids, formaldehyde, methanol, phenolic compounds, and amino acids”: is this specific of bacteria in clouds? All bacteria carry at least some of the functions listed so this is a bit misleading.

L70: “the bacteria need to be metabolically active to biodegrade organic compounds” is a pleonasm

L74: “mimicking the Puy de Dôme” è “mimicking cloud water chemical composition at puy de Dôme”

L90: “influence”: the fact that a link was found between these parameters does not imply causal relationship. 

L126: “suggested”? is the appropriate word? (Isn’t the isolation of strains factual?)

L128: “Enterobacter is pathogenic”. Are all Enterobacter pathogenic? Have these strains in particular been tested?

L 142: what sampling method was used? Give more detail about the isolation of these strains.

L137, 214 and elsewhere: MSA in not a carboxylic acid. Use relevant terms. In addition, provide a reference for its concentration in cloud water (I did not find mention of MSA in the papers cited).

L238: “Control experiments were performed using solutions that contained carboxylic acids but no bacterial cells.”: were this carried out for both light and dark conditions?

L247: “with normal functioning cells usually maintaining a constant ADP/ATP ratio”: what is normal functioning? And this statement requires a reference.

L249: Specify what are ADP and ATP molecules, why these are important in biological systems, and how their ratio relates with metabolic activity.

L281: “Both strains will likely not survive in pH < 4 cloud water during the daytime and nighttime”. There should be a mention to time here. What is the timescale considered? what is the half-life time at pH > 4 versus < 4?

L295: not “production” of compounds, but “release”, since these are from cell lysis.

Figure 3: Why have 2 distinct X axes? They seem to correspond directly to each other.

L351-359: It would be interesting to discuss more about the discrepancy between the compounds released by cells during lysis and their composition: what are the proportions expected of the different categories of compounds (i.e. what are these proportions in the cellular material)?

L365: Again, MSA in not a carboxylic acid.

L463-482: this not a conclusion but a summary.

L 487: “Results from this study imply that there is a minimum cloud water pH threshold at which the bacteria will survive and thrive in during the daytime and/or nighttime”. What is that threshold? The data does not show the existence of a threshold per se, so this is a bit overstated.