the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 10 Mar 2026
Ice-nucleating Bacteria Enriched in the Sea-Surface Microlayer as Potential Sources of Atmospheric Ice-Nucleating Particles
Abstract. The sea-surface microlayer (SML) forms the ocean’s thin biological and chemical film that mediates air-sea exchange. This interface is enriched in surface-active organic matter and biogenic particles that can act as ice-nucleating particles (INP), yet the microbial sources of marine INPs remain poorly resolved. Here, we isolated and characterized ice-nucleating bacteria from the SML and underlying water (UW) of a semi-enclosed coastal inlet in Japan. Among 92 bacterial isolates, six strains induced freezing above –15 °C, indicating active ice nucleation. These isolates, affiliated with Flavobacteriia and Gammaproteobacteria, were heat-labile, and lost activity after 0.22 μm filtration, consistent with large (>100 kDa) outer-membrane ice-nucleation proteins. Flow-cytometric assays confirmed that heating up to 100 °C did not cause a decline in bacterial cell numbers, indicating that INA loss resulted from denaturation of heat-labile proteins rather than cell lysis. In contrast, Proteinase K treatment caused marked membrane disruption, suggesting that proteolytic inactivation and cell damage jointly contributed to INA reduction. One strain, 4U17, retained high INA after Proteinase K treatment despite extensive cell lysis, suggesting a protease-resistant nucleator that may be non-proteinaceous or shielded within extracellular or cell-derived particles. Amplicon sequencing of environmental samples revealed that taxa related to these ice-nucleating bacteria were consistently enriched in the SML relative to UW. Together, our results identify the marine SML as a reservoir for biogenic ice nucleators that may contribute to the oceanic source of atmospheric INPs.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2026-118', Anonymous Referee #1, 05 Jun 2026
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AC1: 'Reply on RC1', Shu-Kuan Wong, 28 Jul 2026
We thank the reviewer for the detailed and constructive assessment. We address the general comments, specific comments, and technical corrections below.
A) General comments:
Comment 1 (sampling information). The manuscript lacks a clear description of when samples were collected, how they were stored prior to analyses, and whether the isolated strains and amplicon sequencing data originated from the same samples.
Response: We have clarified the provenance of the two datasets. Both the ice-nucleating isolates and the 16S rRNA amplicon data derive from the same water samples collected during the December 2012 campaign at Aburatsubo Inlet (the amplicon data published in Wong et al., 2021). The two datasets are therefore directly paired rather than merely from the same site. Plating for isolation of bacteria was done immediately after sampling. Water samples were also filtered immediately after sampling and stored at -20C until DNA extraction. The Methods now state this explicitly, together with sample handling (Lines 120-122) .
Comment 2 (flow cytometry / SYBR Green I). SYBR Green I is membrane-permeant and stains intact and ruptured cells alike, so it cannot discriminate intact from compromised cells; the conclusion that membranes “remained intact” after heating is not supported, and the reduced counts after Proteinase K may be a flow-cytometric/thresholding artifact.
Response: We agree. SYBR Green I stains nucleic acids in both intact and membrane-compromised cells, so our measurements represent total cell counts rather than a live/dead or membrane-integrity readout. We have revised the manuscript throughout to reflect this.
We did not repeat the assay with dual SYBR/PI staining. Accordingly, we now treat the flow-cytometric data purely as total-cell counts and no longer draw any inference about cell state or membrane integrity from them. For strains 5M3, 5D9, and 5M29 at 50°C, total cell counts were comparable to the unheated 4°C control (within 0.88–1.04; Table 2) while ice-nucleation activity was nonetheless lost, indicating that the loss of activity at 50°C was not caused by a loss of countable cells and is more consistent with denaturation of a heat-labile component. At 100°C, counts likewise remained close to the control for most strains; because SYBR Green I enumeration reports total cell abundance rather than integrity, we do not interpret these counts as evidence for or against a change in cell state at either temperature. We also agree regarding the Proteinase K counts: their reduction may reflect released DNA falling below the fluorescence threshold, aggregation, a visible precipitate that formed after enzyme addition, or removal of aggregated material during the 30 µm pre-filtration. We therefore no longer interpret the Proteinase K counts as evidence of membrane disruption; the proteinaceous nature of the nucleators is supported by the heat-treatment and Proteinase K results together, with the Proteinase K counts presented as consistent but not decisive.
Comment 3 (Supplementary Table 1). The authors state that 92 strains were tested and refer to Supplementary Table 1, but no supplementary information was found.
Response: We apologise for the omission. Supplementary Table 1, listing all 92 isolates screened for ice-nucleation activity, is now included with the revised submission.
Comment 4 (statistics). The pairwise Student’s t-test should be justified against the assumption of normality; the ice-nucleation data may not be normally distributed, in which case a non-parametric approach would be more suitable, and for three or more dependent groups an appropriate omnibus test with post-hoc comparisons should be used. Please also clarify which metric was used for the statistical comparisons.
Response: We agree that the paired Student’s t-test was not appropriate given the small sample size (n = 8 droplets) and the likely non-normality of the freezing-temperature data. We now use non-parametric tests throughout, performed on the per-droplet freezing temperatures of the eight replicate droplets per strain and treatment. Each droplet’s freezing temperature was taken as the warmest step at which it was scored frozen. The three heat conditions (control, 50°C, 100°C) were compared by the Kruskal–Wallis test with Holm-corrected Wilcoxon rank-sum post-hoc tests; the Proteinase K, 0.22 µm filtration, and 100 kDa size-fractionation treatments were each compared with their control by the Wilcoxon rank-sum test. Re-analysis changed several significance calls, which we have corrected in the text and figures (most notably: 4U19 is no longer significant at 100°C; 5M3 is no longer significant after Proteinase K; and the 0.22 µm filtration effect is significant only for strain 5G21).
B) Specific comments :
Specific comment 1. “Underlying water” / “UW” consistency.
Response: We have defined “underlying water (UW)” at first use and use “UW” consistently thereafter.
Specific comment 2. Number and dates of water samples (p.5, ll.103–106; p.6, ll.122–126).
Response: The collection dates and the number of samples are now given, and the Methods state that the amplicon and isolate data derive from the same water aliquots.
Specific comment 3. Isolate-code first letters (p.7, ll.159–160).
Response: We have corrected and completed the isolate-code key: station (inlet mouth = 4, pier = 5) followed by sample source (G = glass plate, D = drum sampler, M = polycarbonate membrane for SML; U = underlying water) and isolate number.
Specific comment 4. 10⁶ cells mL⁻¹ threshold (p.8, l.173).
Response: We agree. The value derives from a single ice-nucleating strain of Pseudomonas syringae and was not intended as a general threshold, so we have revised the text to attribute it explicitly. We now also report that our working densities (OD₆₀₀ 0.2–0.6; 10⁷–10⁸ cells mL⁻¹) were determined from calibration curves constructed with 20 representative isolates using DAPI direct counts, and are comparable to or higher than those used in large-scale ice-nucleation screening (Failor et al., 2017: OD₆₀₀ 0.2 ≈ 1.3 × 10⁸ CFU mL⁻¹). Sufficiency for the six characterised isolates is in any case demonstrated empirically, as all six nucleated ice at −10 °C or warmer at these densities, and every treatment comparison is internal to a single cell suspension, so our conclusions do not depend on an absolute threshold. We acknowledge that per-cell nucleation frequency varies by orders of magnitude among taxa, and have therefore qualified strains scored as INA-negative as showing no detectable activity at the densities and temperatures tested.
Specific comment 5. 8 vs 16 droplets (p.8, ll.175–176).
Response: We have reconciled this: two sets of eight droplets (16 in total) were dispensed per strain, and the ≥50% criterion (≥4 of 8) was applied to each set. The Methods and Results now use consistent wording.
Specific comment 6. Reference for 4°C/2 h incubation (p.8, ll.178–179).
Response: The original statement asserted a mechanism ("to promote the expression of ice nucleation proteins") that our experiments do not test, so we have reworded it to describe only the procedure performed and cited an established droplet-freezing protocol that includes an equivalent cold pre-incubation step: "The PCR tubes were incubated at 4 ℃ for 1 h before the assay, following the cold-preincubation step used in bacterial droplet-freezing protocols (Failor et al., 2017)." We also take this opportunity to correct an error in the original manuscript: the pre-assay incubation was 1 h, not 2 h as stated, and we have corrected this consistently throughout the Methods.
Specific comment 7. “IN factor” vs “INA factor”; INA terminology (p.9, l.195; p.17, ll.411–414).
Response: We have standardised terminology: INA denotes ice-nucleation activity throughout, “INA bacteria” / “INA-positive isolates” denote the organisms, and the inconsistent “IN/INA factor” usage has been removed.
Specific comment 8. Proteinase K incubation conditions (p.9, ll.195–198).
Response: The incubation temperature and duration are now specified and made consistent between the Methods and the Table 2 caption.
Specific comment 9. “4°C control” vs “unheated control” (p.10, l.228).
Response: These refer to the same control; we now use “unheated control” consistently.
Specific comment 10. Retentate (>100 kDa) recovery (p.10, l.243).
Response: We now describe how the retentate was recovered and resuspended in sterile 1× PBS before assay (Line 216).
Specific comment 11. Table 1 caption grammar.
Response: Corrected to “six potentially ice-nucleating bacterial isolates.”
Specific comment 12. Final freezing temperature unchanged for 4U19, 5M3, 5G21 (p.12, ll.272–274).
Response: The reviewer is correct. Re-examination of the data confirms that the final freezing temperature was largely unchanged only for 4U19 (whose untreated final freezing was already low); it decreased in 5M3 and 5G21. We have rewritten the sentence accordingly (Line 257-259).
Specific comment 13. Statistical test for Table 2 / Fig. legends (p.12, l.291).
Response: The test is now specified throughout (see General Comment 4).
Specific comment 14. Table 2 normalisation.
Response: We now note that Table 2 counts derive from cultures prepared independently for flow cytometry and do not correspond to the specific suspensions used in the droplet-freezing assays; replicate normalisation is described in the caption.
Specific comment 15. Supporting references (p.14, ll.337–340; p.16, ll.387–389; p.19, ll.454–456).
Response: Supporting references have been added to the indicated statements.
Specific comment 16. “Droplets freezing completely at –20°C” (p.15, ll.344–345).
Response: Reworded to “all droplets had frozen by –20°C.” (Line 317)
Specific comment 17. Type I/II/III classification (p.16, ll.384–387).
Response: We now attribute Type I–III to classes of ice-nucleation structure/protein aggregation state rather than to bacterial taxa (Turner et al., 1990, 1991) (Lines 351-352), and introduce this framework earlier in the Introduction (Lines 85-86).
Specific comment 18. “Other bacterial lineages” (p.16, ll.390–392).
Response: We have named the taxa concerned (Lines 359-361). The revised text specifies Flavobacterium, Psychrobacter, Sphingomonas and Exiguobacterium from Siberian permafrost (Ponder et al., 2005), and Xanthomonas, Stenotrophomonas, Luteimonas, Acinetobacter, Bacillus and Microbacterium from snow (Mortazavi et al., 2008), and notes that in both studies activity occurred at colder temperatures and with lower per-cell efficiency than in the classical Gammaproteobacteria. We have also clarified our novelty claim based on the report of ice nucleation in Flavobacterium (Ponder et al., 2005) which shows that the capacity is not unprecedented within the Flavobacteriia, while the genera reported here have not previously been linked to ice nucleation.
Specific comment 19. Taxonomic identities in Beall et al. (2021) sentence (p.19, ll.458–460).
Response: The relevant taxa are now named to improve clarity (Lines 419-420).
C) Technical corrections:
Response: We thank the reviewer for these corrections. All listed items have been implemented: films→film; “a major contributors”→“major contributors”; atmopsheric→atmospheric; intristic→intrinsic; “a relatively warm”→“relatively warm”; clam→calm; theat→that; length16S→length 16S; capitalisation of “Subsequently”; ml⁻¹→mL⁻¹ throughout; “the”→“then”; “are”→“is”; removal of “(IN)” and “(INB)”; “INA-active”→“INA”; “INA activity”→“ice-nucleation activity”; “underlying water”→“UW” at the indicated lines; “sea-spray aerosols (SSA)”→“SSA” where already defined; and the Fig. 6 caption “5M9”→“5M29”.
Citation: https://doi.org/10.5194/egusphere-2026-118-AC1
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AC1: 'Reply on RC1', Shu-Kuan Wong, 28 Jul 2026
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RC2: 'Comment on egusphere-2026-118', Anonymous Referee #2, 19 Jul 2026
Review on Wong et al. with the title ‘Ice-nucleating Bacteria Enriched in the Sea-Surface Microlayer as Potential Sources of Atmospheric Ice-Nucleating Particles’
Exchange processes between ocean and atmosphere are very important and gaining more recognition within the last 15 years. The biological ice nucleating particles enriched in the sea surface microlayer, emitted to the atmosphere and being transported to mixed phase cloud level, might be important components for inducing cloud glaciation and precipitation formation. The study of Wong et al. shows extensive work to study the biological taxa and ice nucleation activity of almost 100 bacterial isolates in the sea microlayer and underlying water at the coast of Japan and contribute therefore to this overarching theme. Despite of taxonomic analysis, sample cultivation, flow cytometry and freezing analysis using assay technique, different physical (filtration, heating) and chemical treatments (Proteinase K for cell membrane lysis and protein breakdown) haven been done to narrow down properties of ice nucleation agents. The main results of Wong et al. is that they found enrichment of ice active bacteria (6 out of 92) in the SML at the coast of Japan, affiliated with Flavobacteriia and Gammaproteobacteria, likely caused by membrane ice-nucleation proteins. One strain behaved differently and retained its ice activity after treatments, which was interpreted as non-proteinaceous or shielded nucleators.
As I am not a biologist I will focus on reviewing the ice nucleation part. The comprehensive study of Wong et al. is well written and based on solid concept, despite of the approach of comparing frozen fraction rather than concentrations of ice nucleating material. The study of Wong et al. offers scientific value which is in the scope of the journal Biogeosciences after revising some shortcomings, which will be addressed in the following, I recommend the study for publication.
It has been known for a long time that specific proteins are responsible for the ice nucleation activity of bacteria found in terrestrial or plant related environments such as Pseudomonas syringae with different properties described by different types of ice activity (Wolber et al., 1986; Orser et al., 1985, Yankofsky et al., 1981, Turner et al, 1990, Turner et al., 1991). Later, more details become clear about the relation of ice activity and protein molecular length, structure as well as oligomerization state of ice active proteins from InaZ and the role of cell membranes (Lukas et al., 2020; Schwidetzky et al., 2021; Forbes et al., 2022; Lukas, et al., 2022; Hartmann et al., 2022).
Further, also other macromolecules such as polysaccharides (Pummer et al., 2012 and 2015), also in the marine environment (Alpert et al., 2022; Hartmann et al., 2025) and also other microorganism (algae, archeae, fungi, etc.) as bacteria are important source of ice nucleators from the ocean (e.g., Creamean et al., 2021; Xi et al., 2021; Hartmann et al., 2025). Recently, it has been shown that previously unrecognized class of fungal ice-nucleating proteins have bacterial ancestry (Eufemio et al., 2026). The study of Wong et al, would benefit from a thorough research and a comprehensive overview of the current state of art regarding bacterial ice nucleation proteins, current knowledge about ice nucleation active microbial candidates and other ice active macromolecules in the sea microlayer. A comparison or discussion in the context with similar studies at coasts would be beneficial (e.g., Castenschiold). Another aspect which needs to be discussed as Wong et al, sampled at the coast, that there might also be some influence by terrestrial runoff which needs further discussion in the manuscript (Schmidt et al., 2026; Wieber et al., 2025; Knackstedt et al., 2018).
Another major point of criticism is the application of frozen fractions for comparing ice activity in general. If exact the same sample with the identical number of ice nucleators is considered a direct comparison is possible. But if different samples from different bacteria are compared it can be that different number of bacteria cells are compared which lead to wrong impressions. For example, if the cultivation was less successful for a certain bacterial strain, i.e., less bacterial cells are produced also the likelihood that ice active proteins are formed is much lower and consequently the observed freezing temperatures are much lower. Just because different concentrations were compared. It is much better to follow the concept of ice nucleator concentration. In Wong et al, to normalize the frozen fraction to cell number might be a doable approach. Further when comparing frozen fractions, it is more difficult to understand in which mode of ice nucleation activity (type I, II or II according to Turner et al., 2090, 1991) is observed.
The manuscript would benefit not only from a comprehensive introduction, but also from an early mention of these various types of protein ice activity (discussion is too late). Further the different treatments to the samples would be better motivated when the underlying research question or hypothesis would be given in the beginning.
Minor points:
For ice nucleation experiments it is always very important to show the lower limit of the freezing measurements. In Wong et al., if I have not overseen anything, just one PBS measurement is presented as lower limit. Depending on PBS solute concentration also freezing point depression can play a role and influence freezing temperatures of the different samples. This as well as potential measurement uncertainties needs to be shown and discussed. Furthermore, it is difficult to reconstruct whether PBS can be considered a handling blank for all samples, or whether the various treatments might have altered the concentration of dissolved substances, e.g. by adding Proteinase K. The reviewer strongly suggests to add some more test showing the lower limit of freezing experiments according to the respective treatments.
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Citation: https://doi.org/10.5194/egusphere-2026-118-RC2 -
AC2: 'Reply on RC2', Shu-Kuan Wong, 28 Jul 2026
We thank the reviewer for the expert assessment of the ice-nucleation aspects of the study and for the detailed literature suggestions, which we have incorporated.
Comment 1 (introduction / current state of the art). The study would benefit from a thorough overview of current knowledge on bacterial ice-nucleation proteins, ice-active microbial candidates, and other ice-active macromolecules in the sea microlayer, including an early mention of the types of protein ice activity, and a comparison with similar coastal studies.
Response: We have expanded the Introduction with two paragraphs: one summarising the molecular basis and structural classes of bacterial ice-nucleation proteins (Wolber et al., 1986; Orser et al., 1985; Yankofsky et al., 1981; Turner et al., 1990, 1991; Lukas et al., 2020, 2022; Schwidetzky et al., 2021; Forbes et al., 2022; Hartmann et al., 2022), which now introduces the Type I–III framework early; and one covering other marine ice nucleators which are polysaccharides and water-soluble macromolecules (Pummer et al., 2012, 2015; Alpert et al., 2022; Hartmann et al., 2025) and algae, archaea, and fungi (Creamean et al., 2021; Xi et al., 2021; Eufemio et al., 2026), and linking SML/precipitation ice nucleators to microbial communities (Beall et al., 2021; Castenschiold et al., 2025).
Comment 2 (terrestrial runoff). As sampling was at the coast, terrestrial runoff may contribute and should be discussed.
Response: We have added a statement to the Discussion acknowledging that, given the semi-enclosed coastal setting, a contribution of land-derived ice nucleators via terrestrial runoff cannot be excluded (Wieber et al., 2025; Schmidt et al., 2026) (Lines 428-429) .
Comment 3 (frozen fraction vs nucleator concentration). Comparing frozen fractions across different bacteria can be misleading if different cell numbers are compared; it is better to follow the concept of ice-nucleator concentration, e.g. by normalising to cell number.
Response: We agree that frozen fractions do not, by themselves, separate intrinsic activity from cell concentration. All suspensions for the freezing assays were standardised by optical density (OD₆₀₀ 0.2–0.6, ≥107-108 cells mL⁻¹ at log phase), and every treatment was compared with its own untreated control prepared from the same suspension, so the within-strain treatment effects that form our main conclusions are not driven by differences in cell number. We considered normalising to absolute nucleus concentration, but the cell concentrations of the freezing-assay suspensions were not measured directly, and our flow-cytometric counts come from independently prepared cultures; we therefore did not compute per-cell efficiencies. The Discussion now makes explicit that between-strain differences in freezing temperature should not be read as per-cell efficiency differences, and that treatment comparisons were made within strains.
Comment 4 (types of protein ice activity; research question). The manuscript would benefit from an early mention of the types of protein ice activity, and the treatments would be better motivated if the underlying research question or hypothesis were stated at the beginning.
Response: The Type I–III framework is now introduced in the Introduction (Comment 1), and we have added an explicit hypothesis at the end of the Introduction stating that we expected the SML to harbour ice-nucleating bacteria enriched relative to underlying water, and used heat, proteolytic, and size-fractionation treatments to test whether their activity is proteinaceous and cell-associated.
Comment 5 (lower limit of freezing; blanks). It is important to show the lower limit of the freezing measurements; only one PBS measurement is presented. Freezing-point depression from PBS solutes and measurement uncertainties should be shown and discussed, and it is unclear whether PBS can be considered a handling blank for all treatments, or whether treatments (e.g. adding Proteinase K) altered dissolved-substance concentrations. Additional tests of the lower limit per treatment are suggested.
Response: We agree this is important, and we can clarify that the design already addresses it. A 1× PBS blank was prepared and processed identically to the isolates in every assay, so each treatment — heat, Proteinase K, 0.22 µm filtration, and 100 kDa ultrafiltration, had its own matched background control that experienced the same handling and medium changes (including the enzyme and buffer added for Proteinase K). The complete-freezing temperature of the blank differed among treatments (e.g. –22°C in the initial assay versus –19°C and –24°C for the >100 kDa retentate and <100 kDa filtrate), confirming that treatment-dependent freezing-point depression was captured by these controls. We have revised the Results and Discussion to state that the background limit was measured per treatment and that freezing temperatures approaching the corresponding blank are treated as indistinguishable from background.
Citation: https://doi.org/10.5194/egusphere-2026-118-AC2
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AC2: 'Reply on RC2', Shu-Kuan Wong, 28 Jul 2026
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General comments
This manuscript presents exciting data on ice nucleation-active bacterial strains isolated from the sea surface microlayer in the Sagami Bay of Japan. The authors identify potentially novel ice nucleation-active strains affiliated with Flavobacteriia and Gammaproteobacteria and characterize their ice nucleation activity through heat treatment, Proteinase K treatments, filtration, and size fractionation of the ice-nucleating material. By demonstrating enrichment of taxa related to the isolates in the sea surface microlayer compared to the underlying water using 16S rRNA amplicon sequencing data, the authors highlight the sea surface microlayer as an important reservoir of biogenic ice nucleators that may contribute to atmospheric ice-nucleating particles.
The manuscript is generally well-written with a logical flow, and the motivation, workflow, and reasoning are clearly presented. However, several sections would benefit from additional clarification, as detailed below. In addition, I recommend a thorough read-through to further improve clarity and streamline the manuscript.
The authors generally do a good job describing the sampling methods. However, it lacks a clear description of when the samples were collected, how the samples were stored prior to analyses, and whether the isolated strains and amplicon sequencing data originated from the same samples or not. Please ensure that this information is clearly presented and easy for the reader to follow.
The authors perform flow cytometry using SYBR Green I Nucleic Acid Gel Stain to assess whether heat treatments (50°C and 100°C) and Proteinase K treatments affect cell integrity. However, to my knowledge, SYBR Green I is generally considered membrane permeable and can enter intact bacterial cells. Consequently, the use of this dye alone does not allow discrimination between intact and ruptured cells. For live/dead discrimination, a combination of dyes such as SYBR Green I and Propidium Iodide (PI) could be used. PI is largely excluded from intact cells and enters primarily cells with compromised membranes.
In this context, the authors report similar cell counts before and after heat treatment at 100°C and conclude that cell membranes largely remained intact. I find this interpretation difficult to support. A more plausible explanation is that, without double-staining, intact and disrupted cells cannot be distinguished, and that this limitation is reflected in the values presented in Table 2. Furthermore, the reduced number of detected events following Proteinase K treatment could result from the flow cytometry analysis itself and because of applied threshold values. Since Proteinase K degrades membrane-associated proteins and may compromise cell envelope integrity, DNA could be released as diffuse fragments that fall below the fluorescence threshold or outside the gating parameters of the flow cytometer, rather than being counted as cellular events.
I therefore suggest that the authors reconsider the purpose of the flow cytometry analysis and the suitability of using SYBR Green I alone for assessing cell integrity in their isolates. If the isolates are still available in stock, it may be possible to repeat the flow cytometry analysis using a dual-staining approach (SYBR Green / PI) for a live/dead assay and distinguish between intact and membrane-compromised cells? Regardless of whether additional experiments are performed, the Methods, Results (including Table 2), and Discussion sections should be revised to ensure that the interpretation of the flow cytometry data is fully supported by the methodology employed.
The authors state that 92 strains were tested for ice nucleation activity and refer to Supplementary Table 1 (page 11, line 249). However, I was unable to find any supplementary information.
Statistical tests were applied to evaluate differences between the control, heat-treated, and Proteinase K-treated samples. The authors use the pairwise Student’s t-test. However, I encourage them to justify the use of this test by assessing whether the data satisfy the assumption of normality (e.g., using the Shapiro-Wilk test). I am concerned that the ice nucleation data may not be normally distributed, in which case a non-parametric approach (Wilcoxon signed rank test) would be more suitable for the data. If three or more dependent groups/treatments (control, 50°C, and 100°C) are compared, a Friedman ANOVA test followed by an appropriate post hoc test may be more suitable. I recommend that the authors evaluate this and include a brief description in the Method section explaining the rationale for their choice of statistical analyses.
In addition, please clarify which metric (e.g., T50) was used for the statistical comparisons.
Specific comments
Throughout the manuscript, please consistently use either “underlying water” or “UW”, and ensure the abbreviation is defined upon first use.
Page 5, lines 103-106: Please clarify the number of water samples collected and the exact dates of collection.
Page 6, lines 122-126: Please specify whether the amplicon sequencing data and INA isolates originate from the same samples. At present, it is unclear whether the UW and SML samples used for these analyses were collected at the same time or on different dates.
Page 7, lines 159-160: The first letter of each sampler abbreviation used in the isolate codes appears to be missing.
Page 8, line 173: Clarify that the minimum of 106 bacterial cells per mL required to detect freezing events at high subzero temperatures is based on the strain of P. syringae studied by Maki et al. (1974). It is unclear whether this assumption applies to other INA bacterial strains, and this should be stated explicitly if generalized.
Page 8, lines 175-176: Could you clarify whether 8 or 16 droplets were used in the droplet freezing assay? The Method section states: “For each strain, eight replicates of 100 μL cell suspension were dispensed into an 8-strip PCR tube (two sets per strain) for analysis”, whereas the Results (page 11, line 254-255) state: “In addition, a threshold of ≥50% frozen droplets (4 of 8 replicates)”.
Page 8, line 178-179: Please provide a reference supporting the statement: “The PCR tubes were incubated at 4℃ for 2 h to promote the expression of ice nucleation proteins”.
Page 9, line 195: The manuscript sometimes uses “IN factor”, other times “INA factor”. Please ensure consistent terminology throughout.
Page 9, line195-198: Please specify the incubation temperature and duration used for Proteinase K treatment.
Page 10, line 228: Does the “4°C control” refer to the “unheated control” described in the previous Method section (page 9, line 204)?
Page 10, line 243: Please clarify how the retentate (>100 kDa) fraction was tested for ice-nucleating activity. Was the material recovered from the filter membrane using sterile 1 x PBS?
Table 1, caption: “six-potential ice-nucleating bacteria isolates” should be “six potentially ice-nucleating bacterial isolates”.
Page 12, lines 272- 274: The statement: Although the onset freezing temperature decreased in all strains after both treatments, the final freezing temperature (the point at which all droplets froze) remained largely unchanged for strains 4U19, 5M3, and 5G21” appears to be supported only for strain 4U19, based on the results shown in Fig. 2. Please clarify.
Page 12, line 291: Please specify what statistical test was used?
Table 2: Why is the initial cell concentration for replicate 2 not normalized to a relative value of 1, as done for replicate 1? While this approach may facilitate within-replicate comparison, it complicates comparison across treatments.
Page 14, lines 337-340: A supporting reference is needed.
Page 15, line 344-345: The statement “with droplets freezing completely at -20°C” should be revised, as droplet either freezes or do not. Please rephrase to indicate that all droplets froze at the respective temperatures.
Page 15, line 363: Please ensure consistent use of the abbreviation “UW”.
Page 16, line 384-387: It would be more accurate to associate Type I, II, and III classifications with ice-nucleating proteins rather than bacteria, as these categories refer to protein types rather than bacterial taxa.
Page 16, lines 387- 389: The statement “Although these bacteria nucleate ice at colder temperatures, they remain relevant to atmospheric processes due to their potential release from marine environments into the lower troposphere” requires a supporting reference.
Page 16: lines 390- 392: “Previous studies have shown that most culturable INA-positive strains from precipitation are Gammaproteobacteria (Failor et al., 2017), though INA activity has also been reported among other bacterial lineages (Ponder et al., 2005; Mortazavi et al., 2008)”.
Please specify which other bacterial lineages are referred to here?
Page 17, line 411 and 414: “INA” is defined here as ice-nucleation activity, but this abbreviation has not been previously introduced. Earlier in the manuscript “INA” has been referred to as “ice nucleation-active”. Please ensure consistent terminology throughout.
Page 19, lines 454- 456: A reference is needed to support the following sentence: “Once airborne, such cells or fragments could act as ice nuclei, initiating freezing in mixed-phase clouds at relatively warm subzero temperatures”.
Page 19, lines 458-460: The statement regarding halotolerant ice-nucleating microbes in coastal precipitation would benefit from inclusion of the taxonomic identities of the organisms discussed, to improve clarity for the reader: “The detection of halotolerant, ice-nucleating microbes in coastal precipitation (Beall et al., 2021) supports the idea that marine-derived bacteria, including those reported here, can survive atmospheric transport and influence cloud glaciation”.
Technical corrections
Page 2, line 32: “INA” has not been defined previously.
Page 3, line 54: “films” should be “film”.
Page 3, line 62. “underlying water” is introduced for the first time here. Please define the abbreviation “UW” and ensure consistency.
Page 3, line 67: “a major contributors” should be “a major contributor” or “major contributors”, depending on intended meaning.
Page 4: line 81: “atmopsheric” -> “atmospheric”.
Page 4: line 87: “intristic” -> “intrinsic”.
Page 4: line 90: “a relatively warm subzero temperatures” -> “relatively warm subzero temperatures”
Page 4, line 93: “underlying water” -> “UW”.
Page 5, line 96: “underlying water” -> “UW”.
Page 5, line 106: “clam” -> “calm”.
Page 7, line 145: “length16S” -> “length 16S”.
Page 7, line 165: “subsequently” -> “Subsequently”.
Page 9, line 212: “ml-1” -> “mL-1”.
Page 10, line 240: “the” should be “then”.
Page 11, line 255: “are” -> “is”
Page 14, line 332: remove “(IN)”.
Page 15, line 358: “INA-active strains” -> “INA strains”.
Page 15, line 364: “INA bacteria isolates” -> “INA bacterial isolates”.
Page 16, line 383: Remove the abbreviation “(INB)” as it is not used elsewhere in the manuscript.
Page 16, line 392: “INA activity” should be “ice nucleation activity” or “IN activity”.
Page 18, line 417: “INA activity” -> “ice nucleation activity” or “IN activity”.
Page 18, line 437: “INA activity” -> “ice nucleation activity” or “IN activity”.
Page 18, line 439: “INA-active” should just be “INA”.
Page 19, line 444: “INA-active” should just be “INA”.
Page 19, line 454: “sea-spray aerosols (SSA)” -> “SSA” as it has already been defined earlier in the manuscript.