Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest

Proske, Ulrike; Adams, Michael P.; Porter, Grace C. E.; Holden, Mark; Bäck, Jaana; Murray, Benjamin J.

doi:https://doi.org/10.5194/egusphere-2023-2780

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https://doi.org/10.5194/egusphere-2023-2780

Preprints

04 Jan 2024

| 04 Jan 2024

Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest

Ulrike Proske, Michael P. Adams, Grace C. E. Porter, Mark Holden, Jaana Bäck, and Benjamin J. Murray

Abstract. Ice nucleating particles (INPs) facilitate heterogeneous freezing of cloud droplets and thus modify cloud properties. Hence, it is important to understand the sources of INPs. During the HyICE-2018 campaign, which took place in the boreal forest of Hyytiälä, substantial concentrations of biological INPs were observed despite many potential biological sources of INPs being snow covered. A potential source of INP that were not covered in snow were lichens that grow on trees, hence we investigated these lichens as a potential source of biological INPs in this boreal forest environment. INPs derived from lichen sampled during HyICE-2018 are shown to nucleate ice at temperatures as warm as -5 °C in aqueous solutions extracted from 0.03 g ml^-1 lichen. In particular, we identify two species of INPs active at -16 and -18 °C and smaller than 2 µm in size, which makes them good candidates for atmospheric INPs. These particles are smaller than whole lichen spores. In species specific tests we show that the four investigated lichen species harbour differently sized INPs, which might suggest that some species of lichen are more important as a source of INPs than others. This study shows that lichen from a European Boreal forest harbour INPs, which may be especially important in habitats where few other biological INP sources are present, such as in a snow covered environment. The great terrestrial abundance of lichen INPs calls for further research to combine their ice nucleating ability with dispersal studies to evaluate the flux of lichenous INPs into the atmosphere as well as to what extent these particles reach heights and locations where they might influence cloud properties.

Received: 27 Nov 2023 – Discussion started: 04 Jan 2024

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27 Jan 2025

Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest

Ulrike Proske, Michael P. Adams, Grace C. E. Porter, Mark A. Holden, Jaana Bäck, and Benjamin J. Murray

Atmos. Chem. Phys., 25, 979–995, https://doi.org/10.5194/acp-25-979-2025,https://doi.org/10.5194/acp-25-979-2025, 2025

Short summary

Ulrike Proske, Michael P. Adams, Grace C. E. Porter, Mark Holden, Jaana Bäck, and Benjamin J. Murray

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-2780', Anonymous Referee #1, 13 Jan 2024

The work by Proske et al. investigates the role of lichen IN from a borreal forest as biological INPs. The authors investigate four different lichen species and state in their introduction that ...While considerable attention has been paid to INPs of bacterial origin, there has been comparably little interest in the ice-nucleating ability of particles that stem from lichen.
However, a quick google scholar search revealed that this statement is incorrect. In fact, a study by Eufemio et al. 2023, https://doi.org/10.5194/bg-20-2805-2023 investigated Lichen as potential INPs in great detail. It is puzzling how the authors have overlooked this study as the topic is identical to their study. In fact, of the four investigated lichen species in this manuscript, three were already reported by Eufemio et al. The samples were also derived from similar snow-covered ecosystems. Eufemio et al. had also already done tests for heat stability and freeze-thaw cycles. In light of that manuscript, the current study does not add any new information to the topic besides confirming the results by Eufemio et al.
In addition, the current work has additional flawes in their manuscript that should be addressed.
The study puts emphasis on the potential size dependency of the lichen INP. Yet again an important study on this topic seem to have been overlooked as Kieft und Ruscetti 1992 https://doi.org/10.1016/0011-2240(92)90042-Z used gamma radiation to determine size of the INs.
- How did the authors determined which lichen species they investigated? visual inspection? genetic testing?
- How did the authors ensure that the lichen were not contaminated with e.g. bacteria on top of the lichen. Any washing steps prior to analysis?
- Lichen is a symbiosis of more then two partners, the idea that is only two is outdated and has been disproven
- p.3 l. 76, the Reference Schwidetzky et al. 2023 PNAS would be fitting as it is the first study that provides conclusive evidence that fungal IN are proteins

Citation: https://doi.org/10.5194/egusphere-2023-2780-RC1
- CC1:
  'Reply on RC1', Benjamin Murray, 15 Jan 2024
  
  This is a brief response to the referee’s comment on the Eufernio et al. paper, before we respond to the full review at a later point. We can only apologise for this omission and thank the referee for pointing it out. We only became aware of the Eufemio paper after our paper was published as a discussion. This recent paper should have been discussed in our paper and we had already noted the inclusion of Eufernio et al. as something that we needed to correct in the revised paper. In the revised paper we will discuss our results in comparison to those of Eufernio et al.
  It is important to note that Eufernio et al. published on lichens sampled in Alaska, whereas ours were sampled in Finland. In the context of the HyIce campaign (Schneider et al. (2021), Brasseur et al. (2022)), these results are of particular relevance. Also, we are using the conclusions of our paper in another paper shortly to be submitted on the INP concentration during HyIce, hence the lichen results from Finland are of more direct relevance.
  We maintain that there has been ‘been comparably little interest in the ice-nucleating ability of particles that stem from lichen.’, even with the Eifernio paper. There have been very few studies on the ice nucleating ability of lichens in comparison to bacteria. We were pleased to see the Eufernio et al. paper as it reinforces one of the messages in our paper, i.e. that lichen are a potentially importance source of atmospheric INP.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2780-CC1
  - AC1: 'Reply to Referee 1', Benjamin Murray, 22 May 2024
    
    The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2023-2780/egusphere-2023-2780-AC1-supplement.pdf
    
    Citation: https://doi.org/10.5194/egusphere-2023-2780-AC1
RC2:
'Referee report on "Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest"', Anonymous Referee #2, 06 Feb 2024

The manuscript presents an analysis of the ice forming activity of lichen wash water. Lichen samples growing on Scots pines in Hyytiäla, Finland, were collected, washed, and the ice forming activity of the water was determined using a drop freezing experiment. In addition, the washing water was filtered and subjected to heat-treatment to study the size and nature of the ice nucleating particles.

The manuscript is well written and gives an overview of many previous studies. However, the new experimental work does not appear to go beyond the screening a few samples and the interpretation of results appears speculative. In its current form the manuscript does not present substantial and convincing new data.
Specific comments:
Introduction
Line 79-80: Explain how ice formation might help to change physiological activity and indicate in what way.
Methods
Line 90 ff: Clarify why the approach was to use a mixture of lichen species for heat-treatment and detailed filtering experiments rather than a single species. The approach seems more suitable for an initial screening or a feasibility study.
Line 110 ff. Was the lichen separated from the tree bark?
Line 114: Explain why an attempt was made to mimic sample B.
Line 118 ff., 123ff: Explain why the particle fraction resembling the windblown particles is suspended (or not) in a gentle, wet extraction. What difference can be expected between wind and water extraction? Smaller or larger particles, more or less numerous? With the aim of using this type of measurement to estimate the atmospheric ice formation potential of lichens from the lichen mass, the similarity of particles washed off to wind-blown needs to be clarified.

Does exposure of lichens to Milli-Q water cause lysis?
Section 2.4. Clarify whether samples are first filtered and then heated or vice versa.
Section 2.5.: For brevity, consider referring to previous descriptions and only mention steps specific to the present experiments.

Line 162 ff.: If I understand correctly, the initially added lichen mass did not dissolve in the water. Why was the initially added lichen mass used to normalise the measurements if the lichen mass was not contained in the droplets? Also, filtering removes the lichen mass from the suspension. Justify the use of the initially added lichen mass to normalize data from filtered samples.
Results
Line 171: Clarify how the hypothesis can be confirmed by the present study? Considering that the water samples may not be representative of aerosolized samples (line 124-125) and that both the concentration of lichen particles in the atmosphere and their ice forming capacity need to be known to make this assessment (line 83-84).
The measurement data can possibly be better interpreted if they are presented as fraction frozen instead of as site density normalized to lichen mass. Below is a comparison of the fraction frozen of non-heat-treated, non-diluted samples B and C (data taken from the provided Assets to the manuscript).
Line 177-178: As can be seen in the plot above, there is a clear offset in the fraction frozen (FF) between the unfiltered and the 10µm filtered B sample (blue triangles). They cannot be considered similar.
Line 193: The experimental detection limit makes the comparison of heat-lability at different temperatures questionable. There is no data above -15°C in the heat-treated data that can be compared.

As shown in your Fig.4 c) and d), there seems to be a large offset between original and dilutions of heat-treated samples. Can this be explained?
Line 220: Explain why lower concentrations can be expected for smaller size fractions.
Line 224: The differences between sample B and C (see figure above) are surprisingly large considering all single lichen samples showed similar spectra.

Ideally, the single species experiments should reproduce the results of the mixed sample when their spectra are added together, weighted according to their proportion in the mixed sample. Can the results of the single species experiments be used to deduce what the difference between B and C might have been?
Line 225 ff: Clarify how “more” and “less” heat-labile particles can be explained? Shouldn’t “all” or “none” entities that are ice active at a certain temperature be affected?
Fig. 5: Why were no dilutions measured for heat-treated samples C?
Line 231-232: Unclear where this step can be seen. There is no data for the heat-treated samples C, unfiltered and 10µm at -18°C and a step can also be seen in Fig.5e) for 0.1µm.
Line 232: Define which “characteristics for different sizes” are referred to and clarify how this can be used to infer the state of these INPs.
Line 238: The logarithmic scale used in Fig. 6 could be misleading here. Overlap can be checked more directly in FF plots. The figure above shows a comparison of the FF of sample B and C. It shows that the freezing signal of the two samples differs considerably.

It is striking that sample B, unfiltered produced the same FF as sample C, filtered through 0.1 µm, raising overall doubt on the presented interpretation of size dependence. The data should be replotted as FF and features discussed based on such figures. The n_m -plots obscure the data by scaling and plotting on a logarithmic ordinate.
Line 240: What is considered 1 order of magnitude (small difference) in Fig. 6 corresponds to about 4°C (large difference) in the FF plot above.
Line 253: Indicate which small particles.
Line 257: Given the difficulty of reproducing measurements from sample B and C, if seems highly uncertain whether the result of a single measurement is reliable. All measurement should be repeated several times before they are compared and interpreted. Since droplet freezing experiments are neither time consuming nor expensive, several repetitions of experiments are desirable before conclusions are drawn.
Discussion
Line 262: Can recommendations be given on how to measure the atmospherically relevant ice formation activity of lichens in a comparable way?
Line 264-278: A comparison of “INPs per gram of lichen” between studies in which the lichen material remained in droplets for freezing experiments (ground powder) and experiments with washing water in which the lichen matter is not contained in the water droplets seems incorrect. Explain how this comparison of concentrations can be justified. Consider limiting the comparison to temperatures at which specific features are observed.
Line 290 ff: The reported size of the lichen INPs is questionable as the same freezing curve was measured from sample B, unfiltered and sample C, filtered through 0.1µm filter (see figure above).
Line 304: What is the size of the propagules? Consider adding a definition.
Line 318 f: A correlation to which meteorological variable would provide a strong indication of lichen INP?
Line 327: The decrease in concentration with size indicates that a large fraction of these INPs are larger. Clarify what fraction of the -16°C species is smaller than 0.02µm.
Line 329: This conclusion does not appear to be supported by the data. Figure 4 shows a reduction in all sized after heat treatment.
Line 330: Clarify which characteristics are referred to and explain how they support the suggestion of these two different states.
Line 335-336: Clarify how it can be concluded whether a species is an important source of INP based on the current results without knowing the abundance of lichen particles in the atmosphere.
Line 337ff: Since there is no information on the abundance of such smaller lichen entities in the atmosphere, it is speculation whether they contribute INP. Furthermore, the fact that they may be dissolved in water does not necessarily indicate their presence in the atmosphere.
Line 340ff: As the authors explain below (Line 343ff), the ice activity of lichens without atmospheric concentration data is not evidence that they are a source of atmospheric INPs, and other approaches are necessary to clarify the importance of lichens as INPs.
Technical corrections:
Line 18: Provide a reference to “formation of ice in clouds is amongst one of the least well understood of these processes.”
Line 29-30: References should be in brackets.
Line 47: should it be “Fruticose” instead of “Fructiose”?
Line 123: Clarify what is meant by “metal housing”. The filter holder?
Line 158: Define “EF600”
Line 176: consider introducing sample B1 and B2 in Sect. 2.2
Line 280: ... temperatures in our study.
Line 290: Provide a reference for the smallest lichen spore size of 1µm.

Citation: https://doi.org/10.5194/egusphere-2023-2780-RC2
- AC2: 'Reply to referee 2', Benjamin Murray, 22 May 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2023-2780/egusphere-2023-2780-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2780-AC2

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2023-2780', Anonymous Referee #1, 13 Jan 2024

The work by Proske et al. investigates the role of lichen IN from a borreal forest as biological INPs. The authors investigate four different lichen species and state in their introduction that ...While considerable attention has been paid to INPs of bacterial origin, there has been comparably little interest in the ice-nucleating ability of particles that stem from lichen.
However, a quick google scholar search revealed that this statement is incorrect. In fact, a study by Eufemio et al. 2023, https://doi.org/10.5194/bg-20-2805-2023 investigated Lichen as potential INPs in great detail. It is puzzling how the authors have overlooked this study as the topic is identical to their study. In fact, of the four investigated lichen species in this manuscript, three were already reported by Eufemio et al. The samples were also derived from similar snow-covered ecosystems. Eufemio et al. had also already done tests for heat stability and freeze-thaw cycles. In light of that manuscript, the current study does not add any new information to the topic besides confirming the results by Eufemio et al.
In addition, the current work has additional flawes in their manuscript that should be addressed.
The study puts emphasis on the potential size dependency of the lichen INP. Yet again an important study on this topic seem to have been overlooked as Kieft und Ruscetti 1992 https://doi.org/10.1016/0011-2240(92)90042-Z used gamma radiation to determine size of the INs.
- How did the authors determined which lichen species they investigated? visual inspection? genetic testing?
- How did the authors ensure that the lichen were not contaminated with e.g. bacteria on top of the lichen. Any washing steps prior to analysis?
- Lichen is a symbiosis of more then two partners, the idea that is only two is outdated and has been disproven
- p.3 l. 76, the Reference Schwidetzky et al. 2023 PNAS would be fitting as it is the first study that provides conclusive evidence that fungal IN are proteins

Citation: https://doi.org/10.5194/egusphere-2023-2780-RC1
- CC1:
  'Reply on RC1', Benjamin Murray, 15 Jan 2024
  
  This is a brief response to the referee’s comment on the Eufernio et al. paper, before we respond to the full review at a later point. We can only apologise for this omission and thank the referee for pointing it out. We only became aware of the Eufemio paper after our paper was published as a discussion. This recent paper should have been discussed in our paper and we had already noted the inclusion of Eufernio et al. as something that we needed to correct in the revised paper. In the revised paper we will discuss our results in comparison to those of Eufernio et al.
  It is important to note that Eufernio et al. published on lichens sampled in Alaska, whereas ours were sampled in Finland. In the context of the HyIce campaign (Schneider et al. (2021), Brasseur et al. (2022)), these results are of particular relevance. Also, we are using the conclusions of our paper in another paper shortly to be submitted on the INP concentration during HyIce, hence the lichen results from Finland are of more direct relevance.
  We maintain that there has been ‘been comparably little interest in the ice-nucleating ability of particles that stem from lichen.’, even with the Eifernio paper. There have been very few studies on the ice nucleating ability of lichens in comparison to bacteria. We were pleased to see the Eufernio et al. paper as it reinforces one of the messages in our paper, i.e. that lichen are a potentially importance source of atmospheric INP.
  
  Citation: https://doi.org/10.5194/egusphere-2023-2780-CC1
  - AC1: 'Reply to Referee 1', Benjamin Murray, 22 May 2024
    
    The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2023-2780/egusphere-2023-2780-AC1-supplement.pdf
    
    Citation: https://doi.org/10.5194/egusphere-2023-2780-AC1
RC2:
'Referee report on "Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest"', Anonymous Referee #2, 06 Feb 2024

The manuscript presents an analysis of the ice forming activity of lichen wash water. Lichen samples growing on Scots pines in Hyytiäla, Finland, were collected, washed, and the ice forming activity of the water was determined using a drop freezing experiment. In addition, the washing water was filtered and subjected to heat-treatment to study the size and nature of the ice nucleating particles.

The manuscript is well written and gives an overview of many previous studies. However, the new experimental work does not appear to go beyond the screening a few samples and the interpretation of results appears speculative. In its current form the manuscript does not present substantial and convincing new data.
Specific comments:
Introduction
Line 79-80: Explain how ice formation might help to change physiological activity and indicate in what way.
Methods
Line 90 ff: Clarify why the approach was to use a mixture of lichen species for heat-treatment and detailed filtering experiments rather than a single species. The approach seems more suitable for an initial screening or a feasibility study.
Line 110 ff. Was the lichen separated from the tree bark?
Line 114: Explain why an attempt was made to mimic sample B.
Line 118 ff., 123ff: Explain why the particle fraction resembling the windblown particles is suspended (or not) in a gentle, wet extraction. What difference can be expected between wind and water extraction? Smaller or larger particles, more or less numerous? With the aim of using this type of measurement to estimate the atmospheric ice formation potential of lichens from the lichen mass, the similarity of particles washed off to wind-blown needs to be clarified.

Does exposure of lichens to Milli-Q water cause lysis?
Section 2.4. Clarify whether samples are first filtered and then heated or vice versa.
Section 2.5.: For brevity, consider referring to previous descriptions and only mention steps specific to the present experiments.

Line 162 ff.: If I understand correctly, the initially added lichen mass did not dissolve in the water. Why was the initially added lichen mass used to normalise the measurements if the lichen mass was not contained in the droplets? Also, filtering removes the lichen mass from the suspension. Justify the use of the initially added lichen mass to normalize data from filtered samples.
Results
Line 171: Clarify how the hypothesis can be confirmed by the present study? Considering that the water samples may not be representative of aerosolized samples (line 124-125) and that both the concentration of lichen particles in the atmosphere and their ice forming capacity need to be known to make this assessment (line 83-84).
The measurement data can possibly be better interpreted if they are presented as fraction frozen instead of as site density normalized to lichen mass. Below is a comparison of the fraction frozen of non-heat-treated, non-diluted samples B and C (data taken from the provided Assets to the manuscript).
Line 177-178: As can be seen in the plot above, there is a clear offset in the fraction frozen (FF) between the unfiltered and the 10µm filtered B sample (blue triangles). They cannot be considered similar.
Line 193: The experimental detection limit makes the comparison of heat-lability at different temperatures questionable. There is no data above -15°C in the heat-treated data that can be compared.

As shown in your Fig.4 c) and d), there seems to be a large offset between original and dilutions of heat-treated samples. Can this be explained?
Line 220: Explain why lower concentrations can be expected for smaller size fractions.
Line 224: The differences between sample B and C (see figure above) are surprisingly large considering all single lichen samples showed similar spectra.

Ideally, the single species experiments should reproduce the results of the mixed sample when their spectra are added together, weighted according to their proportion in the mixed sample. Can the results of the single species experiments be used to deduce what the difference between B and C might have been?
Line 225 ff: Clarify how “more” and “less” heat-labile particles can be explained? Shouldn’t “all” or “none” entities that are ice active at a certain temperature be affected?
Fig. 5: Why were no dilutions measured for heat-treated samples C?
Line 231-232: Unclear where this step can be seen. There is no data for the heat-treated samples C, unfiltered and 10µm at -18°C and a step can also be seen in Fig.5e) for 0.1µm.
Line 232: Define which “characteristics for different sizes” are referred to and clarify how this can be used to infer the state of these INPs.
Line 238: The logarithmic scale used in Fig. 6 could be misleading here. Overlap can be checked more directly in FF plots. The figure above shows a comparison of the FF of sample B and C. It shows that the freezing signal of the two samples differs considerably.

It is striking that sample B, unfiltered produced the same FF as sample C, filtered through 0.1 µm, raising overall doubt on the presented interpretation of size dependence. The data should be replotted as FF and features discussed based on such figures. The n_m -plots obscure the data by scaling and plotting on a logarithmic ordinate.
Line 240: What is considered 1 order of magnitude (small difference) in Fig. 6 corresponds to about 4°C (large difference) in the FF plot above.
Line 253: Indicate which small particles.
Line 257: Given the difficulty of reproducing measurements from sample B and C, if seems highly uncertain whether the result of a single measurement is reliable. All measurement should be repeated several times before they are compared and interpreted. Since droplet freezing experiments are neither time consuming nor expensive, several repetitions of experiments are desirable before conclusions are drawn.
Discussion
Line 262: Can recommendations be given on how to measure the atmospherically relevant ice formation activity of lichens in a comparable way?
Line 264-278: A comparison of “INPs per gram of lichen” between studies in which the lichen material remained in droplets for freezing experiments (ground powder) and experiments with washing water in which the lichen matter is not contained in the water droplets seems incorrect. Explain how this comparison of concentrations can be justified. Consider limiting the comparison to temperatures at which specific features are observed.
Line 290 ff: The reported size of the lichen INPs is questionable as the same freezing curve was measured from sample B, unfiltered and sample C, filtered through 0.1µm filter (see figure above).
Line 304: What is the size of the propagules? Consider adding a definition.
Line 318 f: A correlation to which meteorological variable would provide a strong indication of lichen INP?
Line 327: The decrease in concentration with size indicates that a large fraction of these INPs are larger. Clarify what fraction of the -16°C species is smaller than 0.02µm.
Line 329: This conclusion does not appear to be supported by the data. Figure 4 shows a reduction in all sized after heat treatment.
Line 330: Clarify which characteristics are referred to and explain how they support the suggestion of these two different states.
Line 335-336: Clarify how it can be concluded whether a species is an important source of INP based on the current results without knowing the abundance of lichen particles in the atmosphere.
Line 337ff: Since there is no information on the abundance of such smaller lichen entities in the atmosphere, it is speculation whether they contribute INP. Furthermore, the fact that they may be dissolved in water does not necessarily indicate their presence in the atmosphere.
Line 340ff: As the authors explain below (Line 343ff), the ice activity of lichens without atmospheric concentration data is not evidence that they are a source of atmospheric INPs, and other approaches are necessary to clarify the importance of lichens as INPs.
Technical corrections:
Line 18: Provide a reference to “formation of ice in clouds is amongst one of the least well understood of these processes.”
Line 29-30: References should be in brackets.
Line 47: should it be “Fruticose” instead of “Fructiose”?
Line 123: Clarify what is meant by “metal housing”. The filter holder?
Line 158: Define “EF600”
Line 176: consider introducing sample B1 and B2 in Sect. 2.2
Line 280: ... temperatures in our study.
Line 290: Provide a reference for the smallest lichen spore size of 1µm.

Citation: https://doi.org/10.5194/egusphere-2023-2780-RC2
- AC2: 'Reply to referee 2', Benjamin Murray, 22 May 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2023-2780/egusphere-2023-2780-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2023-2780-AC2

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

AR by Benjamin Murray on behalf of the Authors (23 May 2024) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (17 Jun 2024) by Paul Zieger

RR by Hinrich Grothe (18 Jul 2024)

RR by Anonymous Referee #4 (23 Jul 2024)

Suggestions for revision or reasons for rejection

Proske et al. have revised a manuscript based on INP measurements from several lichen species collected in Finland. The study presents results including size-resolved analysis via filtration and thermal treatments to assess the INP composition of the lichen samples. Although significant effort has been made in the revision process, several major issues remain that need to be addressed before the manuscript can be considered for publication.

General comments:

Abstract: I understand the concern of the previous reviewers regarding the novelty of the work. The interesting aspect is that these lichens are present and consist of warm temperature INPs during winter when other more prominent/abundant biological sources are covered in snow, thus, could be important for influencing the wintertime airborne INP population. This concept should be highlighted more clearly, especially in the abstract, but additionally throughout.

Hypothesis: Shouldn’t this hypothesis be more specific, based on what was actually tested? The study only tests winter samples. I would guess there is some level of dormancy in the winter and the samples might behave differently in the summer. Also, the study does not actually test the hypothesis in that the presence of lichen is not investigated in the airborne INPs from HyICE-2018. Rather, the study tests the potential of local sources to contain INPs that could become airborne during the winter. The hypothesis should be rewritten to reflect what is actually verified/denied in this study and should be revisited in the discussion section.

“Steps”: It is very difficult to visually discern the steps in the current figures. Also, if trying to discuss different INP populations, it is best practice to use differential spectra. A couple of suggestions here: 1) make the current spectra bigger/wider so the small differences are easier to see and 2) make a figure with differential spectra, so that the two populations at -16C and -18C are evident.

Filtrations: Why were the same filtrations not executed on all the samples? Specifically, sample B was not subject to the 0.02 µm as sample C was. For the individual species, only 2 µm filtration was done. It is difficult to intercompare the samples when the same filtrations were not done. The authors could either omit the 0.02 µm results or very clearly describe why it was only done on one sample. Also, why were the same filtrations not done on the individual species? That may have helped determine which are the most dominant INPs in the sample mixtures, by being able to compare similarly-manipulated samples.

Proposed emission mechanisms: First, suspending lichen in a bunch of water would not replicate possible aerosolization methods in the real environment (lines 132-135). Suspension in water and atmospheric emission mechanisms could be completely different. This sentence should be omitted; the authors should instead state that there could be differences in the suspension solution versus what might actually become airborne. Second, the authors discuss how the smaller INPs could become airborne by adhering to large particles. Has this process been evaluated for lichen? What larger particles are present in lichen that these smaller INPs would stick to, that are subject to detaching from the lichen surface? I can see how this emission process is possible for soil surfaces, but it is not clear for lichen. Additional justification for these conclusions should be provided as the link between the results and possible emission processes is currently not clearly drawn.

Reordering text: The results section was hard to follow - it did not flow naturally. I suggest reordering, so that the current section 3.1 is first (describing the individual species) followed by the start of the results up to line 278 (more complex investigation of potentially realistic ambient external mixtures). The text on lines 278-285 and the corresponding figure (comparing the extraction techniques) is more of a methodology testing. I suggest moving this to the methods as it does not test the hypothesis or apply to any sort of emission process that would realistically happen.

Along these lines, I found it unclear that B and C are called “samples” when in reality, they are roughly the same mixture just tested under different suspension techniques. (Side question: Why was one master sample not mixed and then aliquoted for the different extraction methods?) Perhaps just call them the same sample mixture, but in the methods, describe that they were tested differently. You could even call the spectra “hand shaken” and “rotary mixed” or something along those lines.

In general, there are many qualitative comparisons drawn between the results presented here and previous studies, and for inter-sample comparisons (i.e., the use of “steps” or indicating spectral features). It would be a stronger paper if actual concentration values were compared and differential spectra were used for the study sample intercomparisons. And for the filtrations, it would be helpful to present the % of the total INPs that were 10 µm, 2 µm, 0.1 µm, and 0.02 µm.

Discussion section: A few things to point out here.

1. Some of this section belongs in the intro (lines 343-356), as it does not actually help describe or explain the study results.

2. This section would be much more impactful if there was a comparison figure shown, since there are a handful of previous studies evaluating INPs in lichen (something like spectral “ranges” as in Fig 1-10 of Kanji et al. (2017) or a bar plot of spectral ranges as in Fig 4 in Creamean et al. (2021)). It is difficult to put the current results into context without some sort of summarizing visual.

3. Intercomparing onset freezing temperatures with previous studies using different instruments is not recommended. Instrumental limitations and detection limits can yield different ranges of freezing onsets. Try to refrain from onset comparisons unless doing so with just the samples in the current study.

4. The main idea behind this study is to test what local sources might have been observed in the air. Thus, putting these findings into the context of those from the air during HyICE-2018 should be included here. This text basically exists on lines 369-377 in the conclusion section, but I suggest moving it to the discussion.

5. Along these lines, what other sources could be possible during the winter? What about anything on the needle surfaces or resuspension from the snowpack? I realize these were not tested here, but other realistic possibilities should be mentioned and cited.

Specific comments:

Line 3: Specify that these were ambient/airborne INPs, not to be confused with the current work.

Lines 7-8: The abstract does not entirely reflect the actual findings. The authors report on the 0.02 µm results being the most substantial, but filtration was only done on one of the samples. In the paper, several times (e.g., lines 256-257, the discussion, and the conclusions), it is indicated that most of the INPs were in the 0.1-10 µm range. That should be reported here instead.

Lines 44-46: Why is one study reported in onset freezing range and one in median? It is best to compare apples to apples and report median or another common value for both. (Intercomparing onset temperatures between different techniques is not advised; see comment above.)

Lines 120-121: Why stored at room temperature and not at least close to the temperature in which the samples were collected? The authors should provide a statement about the caveats in possible changes to the samples while stored at room temperature. Often, vegetation samples are stored in a refrigerator (something like 4C) or even frozen. The authors cite Stopelli et al. (2014) which describe that certain conditions are known to activate the ice-nucleating activity of bacterial cells. While they are referring to samples in snow water, bacterial activity can change when the temperature of the air increases (i.e., what can happen in the summer compared to winter samples).

Table 1: Why were these particular percentages of species chosen for B and C? There needs to be some justification provided in the text.

Line 320: Why is a quantifiable comparison not possible?

Line 322: Saying they “also” found high variability does not align with the current study. The species presented in Fig 7a look pretty similar. But this is also admittedly subjective. Making this statement more quantifiable, for example by using X orders of magnitude spread at select temperatures, and comparing the same to Eufemio et al. would get the point across, if indeed there was high variability found in both that study and the current one. If not found, why might this study not have “high variability”?

Lines 335-336: What are the typical sizes of whole spore soredia or isidia? Some spores can have sizes down to 2 µm but I am not certain about these two specifically.

Refs:
Creamean, J. M., Ceniceros, J. E., Newman, L., Pace, A. D., Hill, T. C. J., DeMott, P. J., and Rhodes, M. E.: Evaluating the potential for Haloarchaea to serve as ice-nucleating particles, Biogeosciences, 18, 3751–3762, https://doi.org/10.5194/bg-18-3751-2021, 2021.

Kanji, Z. A., Ladino, L. A., Wex, H., Boose, Y., Burkert-Kohn, M., Cziczo, D. J., and Krämer, M.: Overview of Ice Nucleating Particles, Meteorological Monographs, 58, 1.1-1.33, https://doi.org/10.1175/AMSMONOGRAPHS-D-16-0006.1, 2017.

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ED: Reconsider after major revisions (23 Jul 2024) by Paul Zieger

AR by Benjamin Murray on behalf of the Authors (26 Aug 2024) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (30 Aug 2024) by Paul Zieger

RR by Anonymous Referee #4 (20 Sep 2024)

Suggestions for revision or reasons for rejection

Proske et al. have significantly revised the manuscript, improving its clarity and strengthening the conclusions. The manuscript now presents a cohesive narrative, and the new additions have made the findings more robust. This is highly engaging work, and I believe the manuscript is now suitable for publication. I have only a few minor suggestions for the authors to consider before final acceptance.

The novelty of this study lies not only in its location but also in its detailed investigation of INP size. While Kieft and Ruscetti (1990) examined only 0.2 μm INPs, this study expands on that by presenting data from filtration across multiple size ranges. Understanding the size distribution of INPs is undoubtedly valuable information. The authors might consider emphasizing this aspect more explicitly in both the abstract and the conclusion of the introduction.

Line 11: Typo: “Hyytiäläre” should be corrected.

Line 12: Consider adding “previous” or “existing” before “evidence” for clarity.

Line 114: I assume you mean above -24 °C here?

Lines 444-445: The statement, “Many of the lichenous INPs were found to be between 0.1 and 2 μm in size when immersed in water, and those active at temperatures higher than −18 °C were heat-labile,” raises an interesting point. Could some of these particles be bacteria living symbiotically with the lichen? Bacterial cells typically range around ~1 μm and are often heat-labile. While the authors mention that Kieft (1988) did not find cultivable bacteria in lichen samples, it’s important to note that not all ice-nucleating bacteria are easily cultured. The introduction touches on how bacteria, among other things, can contribute to the INPs from lichen samples. Given the size range and heat sensitivity, the authors might want to discuss the potential bacterial connection to their findings, specifically, further in their discussion.

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ED: Publish subject to minor revisions (review by editor) (25 Sep 2024) by Paul Zieger

AR by Benjamin Murray on behalf of the Authors (22 Oct 2024) Author's response Author's tracked changes Manuscript

ED: Publish as is (23 Oct 2024) by Paul Zieger

AR by Benjamin Murray on behalf of the Authors (01 Dec 2024) Manuscript

Journal article(s) based on this preprint

27 Jan 2025

Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest

Ulrike Proske, Michael P. Adams, Grace C. E. Porter, Mark A. Holden, Jaana Bäck, and Benjamin J. Murray

Atmos. Chem. Phys., 25, 979–995, https://doi.org/10.5194/acp-25-979-2025,https://doi.org/10.5194/acp-25-979-2025, 2025

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Ulrike Proske, Michael P. Adams, Grace C. E. Porter, Mark Holden, Jaana Bäck, and Benjamin J. Murray

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Data for the publication "The ice-nucleating activity of lichen sampled in a northern European boreal forest" U. Proske et al. https://doi.org/10.5281/zenodo.8355809

Ulrike Proske, Michael P. Adams, Grace C. E. Porter, Mark Holden, Jaana Bäck, and Benjamin J. Murray

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Ice nucleating particles aid freezing of water droplets in clouds and thus modify clouds' properties. During a campaign in the boreal forest in Finland, substantial concentrations of biological ice nucleating particles were observed, despite many of their potential biological sources being snow covered. We sampled lichen in this location and tested its ice nculeation ability in the laboratory. We find that indeed the lichen harbours INPs, which may be important in such snow covered environments.


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Measurement report: The ice-nucleating activity of lichen sampled in a northern European boreal forest

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