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- Lichen is a symbiosis of more then two partners, the idea that is only two is outdated and has been disproven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

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.