the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
05 Jan 2021
05 Jan 2021
Cultivable, halotolerant ice nucleating bacteria and fungi in coastal precipitation
- 1Scripps Institution of Oceanography, La Jolla, CA, 92037, USA
- 2Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA
- 3Department of Earth and Planetary Sciences, Rutgers University, Piscataway, NJ 08854
- 4Pacific Northwest National Laboratory, Richland, WA, 99354, USA
- 5Department of Atmospheric Science, Colorado State University, Fort Collins, CO, 80523, USA
- These authors contributed equally to this work.
- 1Scripps Institution of Oceanography, La Jolla, CA, 92037, USA
- 2Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA
- 3Department of Earth and Planetary Sciences, Rutgers University, Piscataway, NJ 08854
- 4Pacific Northwest National Laboratory, Richland, WA, 99354, USA
- 5Department of Atmospheric Science, Colorado State University, Fort Collins, CO, 80523, USA
- These authors contributed equally to this work.
Abstract. Ice nucleating particles (INPs) are a rare subset of aerosol particles that initiate cloud droplet freezing at temperatures above the homogenous freezing point of water (−38 °C). Considering that the ocean covers 70 % of the earth's surface and represent a large potential source of INPs, it is imperative that the uncertainties in the identities and emissions of ocean INP become better understood. However, the specific underlying drivers of marine INP emissions and their identities remain largely unknown due to limited observations and the challenge involved in isolating exceptionally rare IN forming particles. By generating nascent sea spray aerosol (SSA) over a range of biological conditions, mesocosm studies show that microbes can contribute to marine INPs. Here, we identify 14 (30 %) cultivable halotolerant ice nucleating microbes and fungi among 47 total isolates recovered from precipitation and aerosol samples collected in coastal air in Southern California. IN isolates collected in coastal air were found to nucleate ice from extremely warm to moderate freezing temperatures (−2.3 to −18 °C). Air mass trajectory analyses, and cultivability in marine growth media indicate marine origins of these isolates. Further phylogenetic analysis confirmed that at least two of the 14 IN isolates were of marine origin. Moreover, results from cell washing experiments demonstrate that most IN isolates maintained freezing activity in the absence of nutrients and cell growth media. This study provides confirmation of previous studies' findings that implicated microbes as a potential source of marine INPs and additionally demonstrates links between precipitation, marine aerosol and IN microbes.
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Charlotte M. Beall et al.
Status: closed
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RC1: 'Comment on acp-2020-1229', Anonymous Referee #2, 20 Jan 2021
General comments:
In this study, the authors collected aerosol and precipitation samples in a coastal environment for INP measurements and cultivation of microorganisms. The microbial isolates from aerosol and precipitation samples were tested for ice nucleation activity. Identification of the isolates was done by DNA sequencing and data base comparison. Phylogenetic analysis, trajectory analysis, and cultivation in marine medium were used to obtain clues for possible marine origin of the isolates. The authors successfully obtained 47 different isolates by cultivation. They found ice nucleation activity in 14 isolates, of which 12 were bacteria and 2 were fungi. Eleven (9 bacteria, 2 fungi) of the 14 IN isolates were derived from precipitation samples and three isolates (bacteria) originated from the aerosol samples. The high fraction of different IN isolates, in particular from the precipitation samples, and number of species with so far unknown IN activity is surprising. Considering the overall limited cultivability of microorganisms under lab conditions, the results of this study indicate that microbial IN activity is far more distributed than previously assumed. The study is suited to the scope of the journal. The method is solid and the results are well presented. The manuscript is well written and well-structured and can be further improved as suggested below.
Specific comments:
Line 25: Better use INP as introduced in line 19 instead of “IN forming particles” as IN is not introduced here and based on IN definition in line 58 it would mean “ice nucleating forming particles”
Line 108: How were the filters pretreated for decontamination before aerosol sampling? Information on blank samples for aerosol sampling and handling should be added.
Line 124: Please add here also the aerosol samples. Moreover, I suggest to add the information given in line 140 about the volume (50 µL) and number of aliquots (30) already here as “microliter aliquots” covers a wide range of possible droplet sizes.
Line 155-157: Please add full information (or a reference) for the performed PCRs (PCR components, concentrations, cycling conditions). Note also, that ribosomal DNA in fungi is 18S and not 16S. Which primers were used for amplification of fungal 18S or did the bacterial primers only coamplify fungal 18S? This part needs some clarification on how the authors obtained fungal 18S sequences. The authors should also clarify and correct this in other parts of the manuscript., e.g., caption table S1, figure S11.
Line 360: As Cryptococcus and Metschikowia are not bacteria but fungi, please change caption to “Identities of 14 …IN bacteria and fungi”. Overall, both fungal species did not receive much attention in the manuscript although the title and abstract raised some expectations. The authors should add some discussion and comparison with the literature for the fungi they advertise in the title.
Line 424: Remove the “sp.” after “syringae” as syringae is the species name.
Line 441: please use “spp.” and not “sp.” if multiple species are meant.
Line 456: “Gammaproteobateria” – typo in bacteria, and missing hyphen (see line 345 Gamma-proteobacteria, be consistent).
Line 465: Lysinibacillus is not gram-negative. Please correct to gram-positive.
Line 490: Can the authors please add some more discussion and more specific suggestions on the “state-of-the art sequencing approaches” they mention here. I wonder how combining INP measurements with state-of the art sequencing should help to identify putative IN microbes that are not recovered by cultivation. The sequencing gives information about composition of the community, which are usually highly diverse, but only a small number of species possesses ice nucleation activity. A diversity analysis, however, does not give information about putative IN abilities of the organisms. Metagenomic (and transcriptomic approaches) are limited by database entries of IN genes, as these genes are not known for the many of the known IN organisms. Also note that some microorganisms (e.g., most known IN fungi) release cell-free IN into the environment. These IN would be covered by the IN measurements but as they do not contain DNA or RNA they would not be covered by the sequencing approaches. Furthermore, without cultivation it seems not feasible to proof the ice nucleation activity of a microorganism, even when (hopefully in future) gene similarities might suggest more candidates.
Table S1: Be consistent - genus and species names should be italics, “sp.” should not; contains several typos in e.g., Bacillaceae, Metschnikowiaceae. Paenibacillus is not a family but a genus, thus it should be Paenibacillaceae in the family column. Column blast identity has an extra comma in line Iso39, missing space in line iso3. IN ability column seems not needed, as IN onset temperature gives the “yes” or “no” information.
Table S4: Genus and species names should be italics, Iso5 – missing space, SSA18 – 7tewartia?
Figure S7: Typo in legend: Metschnikowiaceae; what does the line and the Y? at the right side of the legend mean?
Figure 4 and S10: It is confusing that the orange and yellow triangle symbols (sample 9) described in the legend point to a different direction in the plot. Caption for figure S10 needs to be checked “Sample numbers in the legend indication the precipitation”?
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AC1: 'Reply on RC1', Charlotte Beall, 10 Feb 2021
We thank the anonymous referee for their suggestions and thoughtful comments on how to supplement the discussion of the results. We include their comments and our responses below. Line numbers in our responses refer to the revised manuscript.
Specific comments:
Line 25: “Better use INP as introduced in line 19 instead of “IN forming particles” as IN is not introduced here and based on IN definition in line 58 it would mean “ice nucleating forming particles”
This has been corrected as suggested.
Line 108: “How were the filters pretreated for decontamination before aerosol sampling? Information on blank samples for aerosol sampling and handling should be added.”
Line 110 now reads: “Prior to sampling, filters were pretreated for decontamination by soaking in 10 % H2O2 for 10 minutes and rinsing 3X with ultrapure water.
Background levels of INPs from sampling handling processes were simulated using INP concentrations in aerosol sample field blanks assuming the average sampling volume (2270 L). Simulated INP concentrations across the 3 field blanks ranged between between 0 and 0.1 L-1 at -20 °C (see Fig. S6).”
Line 124: “Please add here also the aerosol samples. Moreover, I suggest to add the information given in line 140 about the volume (50 µL) and number of aliquots (30) already here as “microliter aliquots” covers a wide range of possible droplet sizes.”
Corrected as suggested. Line 129 now reads: “Briefly, the precipitation samples and aerosol sample suspensions were distributed in 24-30 50-microliter aliquots into a clean 96-well disposable polypropylene sample tray.”
Line 155-157: "Please add full information (or a reference) for the performed PCRs (PCR components, concentrations, cycling conditions). Note also, that ribosomal DNA in fungi is 18S and not 16S. Which primers were used for amplification of fungal 18S or did the bacterial primers only coamplify fungal 18S? This part needs some clarification on how the authors obtained fungal 18S sequences. The authors should also clarify and correct this in other parts of the manuscript., e.g., caption table S1, figure S11."
Thank you for bringing this to our attention. The 16S primers were able to capture the fungal 18S sequences and we did not use additional primers. We have added this clarification as well as the PCR reagents and cycling conditions to the manuscript.
Line 163: The PCR reaction contained 0.5 ng ml-1 genomic DNA, 0.2 mM of each primer, and 1x KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2601), and the thermocycler was set to the following program: 95°C for 30 seconds; 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; 72°C for 5 minutes.
Line 170: The 16S primers were able to capture 18S fungal sequences. Primers specific to 18S rRNA were not used.
Caption Table S1 and Fig. S11: 18S fungal sequences were obtained from 16S primers due to coamplification (see Methods Sec. 2.2).
Line 360: "As Cryptococcus and Metschikowia are not bacteria but fungi, please change caption to “Identities of 14 …IN bacteria and fungi”. Overall, both fungal species did not receive much attention in the manuscript although the title and abstract raised some expectations. The authors should add some discussion and comparison with the literature for the fungi they advertise in the title."
Corrected. Table 1’s caption now reads: Identities of 14 cultivable, halotolerant IN bacteria and fungi derived from aerosol or precipitation samples.
We agree that some discussion of fungal INPs is needed given the two fungal IN isolates featured here. Thank you for bringing this to our attention. The following paragraph has been added to Sec. 3.3, Line 391:
Fungal isolates Cryptococcus sp. and Metschikowia sp. represent two new ascomycotic and basidiomycotic IN fungal species, respectively, with INP concentrations 7-8 orders of magnitude lower than the highest reported values for fungal isolates F. armeniacum and F. acuminatum (Kunert et al., 2019). While other IN species of the Ascomycota and Basidiomycota phyla have been previously reported (e.g. Jayaweera and Flanagan, 1982; Kieft et al., 1988; Pouleur et al., 1992), very little is known regarding the distribution and source potential of fungal INPs. Moreover, multiple issues pose challenges to the differentiation of marine vs. terrestrial fungal species (Amend et al., 2019). Many fungi found in the sea are also found in terrestrial environments, and strong correlations with abiotic environmental conditions (Orsi et al., 2013; Tisthammer et al., 2016) and gene expression data (Amend et al., 2012) suggest that some fungi are truly amphibious. Issues with amplicon sequencing pose additional challenges due to coamplification of other eukaryotes and large biases toward terrestrial species in ITS rDNA primers, which were designed using sequence alignments from largely terrestrial representatives (Amend et al., 2019). However, future studies could take advantage of established marine fungi isolation and cultivation techniques to probe the INP source potential of various cultivable marine fungal species (e.g. Kjer et al., 2010; Overy et al., 2019).
Line 424: "Remove the “sp.” after “syringae” as syringae is the species name."
Corrected, thank you.
Line 441: "please use “spp.” and not “sp.” if multiple species are meant."
Corrected. Line 469 now reads: Additionally, whereas (Failor et al., 2017) reports high freezing temperatures between -4 and -12 °C for multiple Pseudomonas spp., none of the Pseudomonas spp. isolated in our study exhibited detectable IN activity.
Line 456: “Gammaproteobateria” – typo in bacteria, and missing hyphen (see line 345 Gamma-proteobacteria, be consistent).
Corrected, thank you.
Line 465: "Lysinibacillus is not gram-negative. Please correct to gram-positive."
Corrected.
Line 490: "Can the authors please add some more discussion and more specific suggestions on the “state-of-the art sequencing approaches” they mention here. I wonder how combining INP measurements with state-of the art sequencing should help to identify putative IN microbes that are not recovered by cultivation. The sequencing gives information about composition of the community, which are usually highly diverse, but only a small number of species possesses ice nucleation activity. A diversity analysis, however, does not give information about putative IN abilities of the organisms. Metagenomic (and transcriptomic approaches) are limited by database entries of IN genes, as these genes are not known for the many of the known IN organisms. Also note that some microorganisms (e.g., most known IN fungi) release cell-free IN into the environment. These IN would be covered by the IN measurements but as they do not contain DNA or RNA they would not be covered by the sequencing approaches. Furthermore, without cultivation it seems not feasible to proof the ice nucleation activity of a microorganism, even when (hopefully in future) gene similarities might suggest more candidates."
We agree that more discussion is needed here to explain how advanced sequencing approaches could help advance understanding of the factors that modulate bio-INP emissions. While it is indeed unlikely that we could identify a single IN species, advanced sequencing methods could illuminate relationships between specific communities and INP freezing activity. For example, high-throughput sequencing techniques for low biomass samples will enable sequencing of individual e.g. 50 μL droplets such that droplet assay measurements of INP concentrations could be related to communities present in low temperature vs high temperature freezing droplets (Minich et al., 2018). The referee also makes a good point about the inability of such methods to identify cell-free INPs. This paragraph has been edited as follows:
Finally, as cultivable populations represent a small fraction of the total microbial community, future studies should combine INP measurements with state-of-the-art sequencing approaches to identify relationships between specific microbial communities and INP freezing activity. Furthermore, a combination of advanced fractionation methods to identify the putative ice nucleating metabolites associated with specific microbial communities and computational networking could illuminate molecular and microbial linkages to ice nucleation and the mechanisms by which the entities work individually or in concert. Further study is also needed to understand the factors, such as atmospheric processing or nutrient limitation, that inhibit or enhance microbe IN behavior, as well as the factors that modulate the emissions of IN bacteria from the ocean surface.
Table S1: "Be consistent - genus and species names should be italics, “sp.” should not; contains several typos in e.g., Bacillaceae, Metschnikowiaceae. Paenibacillus is not a family but a genus, thus it should be Paenibacillaceae in the family column. Column blast identity has an extra comma in line Iso39, missing space in line iso3. IN ability column seems not needed, as IN onset temperature gives the “yes” or “no” information."
Thank you for pointing out the typos. These have been corrected. We agree that the column for IN ability is unnecessary and it has been removed.
Table S4: "Genus and species names should be italics, Iso5 – missing space, SSA18 – 7tewartia?"
Corrected.
Figure S7: "Typo in legend: Metschnikowiaceae; what does the line and the Y? at the right side of the legend mean?"
Corrected, thank you. The symbols are alpha and gamma (to indicate gamma vs alpha-proteobacteria).
Figure 4 and S10: "It is confusing that the orange and yellow triangle symbols (sample 9) described in the legend point to a different direction in the plot. Caption for figure S10 needs to be checked “Sample numbers in the legend indication the precipitation”?"
The triangle orientation has been corrected. (I also found another legend typo for Arthrobacter and Metschnikowia sp. typo and corrected).
The S10 caption typo has been corrected: “Sample numbers in the legend indicate the precipitation or aerosol sample from which the isolate was derived (see Table S3). Datapoints corresponding to isolates from aerosol are outlined in black.”
References:
Minich, J. J., Zhu, Q., Janssen, S., Hendrickson, R., Amir, A., Vetter, R., Hyde, J., Doty, M. M., Stillwell, K., Benardini, J., Kim, J. H., Allen, E. E., Venkateswaran, K. and Knight, R.: KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples, mSystems, 3(3), e00218-17, doi:10.1128/mSystems.00218-17, 2018.
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AC1: 'Reply on RC1', Charlotte Beall, 10 Feb 2021
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RC2: 'Comment on acp-2020-1229', Anonymous Referee #1, 05 Feb 2021
Terrestrial sources of microbial ice nucleators have been the subject of numerous recent investigations to understand their distribution in and meteorological impact to the atmosphere. Since studies to explore their potential marine sources are surprisingly fewer, this is a research are where more information is needed. This study analyzed aerosol and precipitation samples in attempts to enrich for marine microbes that displayed ice nucleation activity. Despite the biases inherent to this approach for, the authors were nevertheless successful in identifying a number of bacterial and fungal isolates that were tested and shown to be active as ice nucleators at temperatures warmer than -18C, and for one, near -2C. The microbiological and ice nucleating properties of the isolates, coupled with their probable sources in the air masses and precipitation samples analyzed, would be of interest to a broad community of researchers interested in marine and atmospheric science.
One general comment is that the results from fourteen isolates with IN activity are summarized in the abstract by presenting the temperature range of activities from -2.3 to -18C. I believe the truly interesting find in this work is the very warm IN temperature for the Brevibacterium strain, as I am not aware of another report of this phenotype in this phylum. My suggestion is to specifically emphasize this in the abstract as this will be a strain that will elicit interest from a range of microbiologists interested in novel mechanisms of biological ice nucleation.
Below are specific points the authors should consider when revising the manuscript:
Lines 32-33; 93-94; 330-332; 462: The phylogenetic information available cannot be used to definitively determine the environmental source of these isolates. The study observations collectively support that the principal aerosol source was marine, but the type and amount of sequence data obtained do not allow, for example, saying that Psychrobacter sp. 1b2 is marine and 2a is not. The phylogenetic resolution possible from the V4 region of the 16S/18S rRNA gene, which is about ~1/3 of the gene sequence, is useful for coarse phylogenetic assignments but is not able to resolve evolutionary relationships between closely related taxa. It is safe to conclude that the major source of cultured microbes was from aerosol samples with a marine origin and that many of the isolates are closely related to marine taxa but resolving environmental source from a few hundred nucleotides of small subunit rRNA sequences is not something that can be done with confidence.
Lines 110-111: Just to confirm that the aerosol samples used for culturing in media with ~35ppt salinity were initially placed in deionized water. It is important to note that this process would represent a significant osmotic shock to the cells. Also, assuming that a “hand” was not literally used for this aseptic procedure, so please clarify how the particles were removed from the filters.
Line 144: Just confirming if it was filtered and then autoclaved. Speculating it could be the reverse because a precipitate typically forms when autoclaving full strength seawater. Please also indicate the source of the seawater.
Line 155 and throughout manuscript: “16S V4 ribosomal DNA fragments were…” This is common lab slang that I suggest rewording here and throughout as "The V4 region of the 16S rRNA gene".
Lines 158-159: Suggest editing to something like “The sequences of the amplified 16S rRNA gene fragments were determined by...”
Lines 159 to 161: Please define acronyms on first use and describe the criteria used for OTU designation.
Line 163: Does this mean that the 16S rRNA gene sequences from different isolates were used to create some type of consensus sequence for each OTU? Please explain this process in more detail.
Lines 167-169: The description of this analysis is confusing to me. Distances >0.1 or 10% in the 16S rRNA gene would represent very large phylogenetic distances and not differences that would be confused at being the same "OTU". Please also indicate the length of DNA sequences used in this comparison.
Lines 206-209: It appears that the cells were washed and then tested immediately after nutrient removal. This would provide no opportunity for the microbes to respond to the experimental conditions, so it is difficult to interpret these results as being relevant to the effect of IN activity on the presence of nutrients.
Line 262: Is each period a separate rainstorm? There is more than one period for some days and with different trajectories in Figure 2, so maybe they are just different sampling periods.
Line 291: Please clarify if the sequence identities in Table S1 are BLAST outputs or based on distance matrices from multiple sequence alignments. Also, a general comment is that a >97% OTU estimate is highly conservative (e.g., Stackebrandt and Ebers 2006, Microbiology Today, 33:152-155).
Line 318-319: The isolated bacterial and fungal taxa cannot easily be compared to microbial communities, which are associations of many many different types of interacting microbes. And if they are Pacific-sourced aerosols, I’m not sure to consider it a "a warmer climate" even though that is the case where they were deposited in SoCal. Can the isolates grow at cold temperatures or is there any other evidence for cold tolerance, if that is in fact what is being implied here? Please revise this sentence for clarity.
Line 334: Is it known if the isolates have optimal growth at salt concentrations in seawater, not just tolerance to the concentrations in the seawater-based media? That would provide physiological support for a marine lifestyle.
Lines 384-385: Does this means than isolates having identical sequences in the portion of the 16S rRNA gene examined were deemed clonal and that one isolate was selected as a representative?
Lines 388-390: Were any of the observations in Figure S11 replicated to confirm that the patterns of IN at these temperatures and isolates were not more affected by the age of cultures or other potential variations in the way the cultures were handled between experiments?
Lines 411-420; 468: Ample time after removal of nutrients may not have been provided in these experiments and the authors should consider limiting this discussion. The one conclusion that can be made is that the activity observed does appear to be associated with the cells and not removed by washing, suggesting the nucleating material is membrane bound or associated with the cell envelope.
Lines 429-432: Please note that the percentage of cells that serve as INPs is temperature dependent, and the "active" fraction values cited from the literature are likely referring to very warm subzero temperatures, whereas for P. syringae populations at temps below -10C, values approaching 100% could be expected. This leads me to suspect the caveats stated are valid for INPs that activate in the -2C range (i.e., Brevibacterium) but may be overly conservative for the colder temperatures of ice nucleation observed in their experiments.
Lines 436-440: The connection being made with the Failor et al. study is ambiguous. Please clarify if the suggestion is that the taxa in the Failor study were of a marine source, that in the isolates in this study were not marine in origin, or something else entirely.
Line 449: I am not able to find where this is discussed further. This suggests some of the IN activities reported were difficult to repeat, which would be consistent with other similar attempts, Failor et al. being one good example.
Line 484-486: General comment to authors: I am most surprised by the fact that out of a group of less than 50, you found one that is active at warmer than -5C and is a member of a phylum where I am not aware of other known examples of this phenotype. In my view, this single isolate may be the most important contribution of this study and will be of interest for additional work to decipher if the mechanism of ice nucleation differs from that of certain Gammaproteobacteria.
Data availability: Please provide database accession information to access the DNA sequence data from this study.
Figure 3: Please indicate the number of aligned nucleotides and method of phylogenetic analysis used for evolutionary tree construction.
Status: closed
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RC1: 'Comment on acp-2020-1229', Anonymous Referee #2, 20 Jan 2021
General comments:
In this study, the authors collected aerosol and precipitation samples in a coastal environment for INP measurements and cultivation of microorganisms. The microbial isolates from aerosol and precipitation samples were tested for ice nucleation activity. Identification of the isolates was done by DNA sequencing and data base comparison. Phylogenetic analysis, trajectory analysis, and cultivation in marine medium were used to obtain clues for possible marine origin of the isolates. The authors successfully obtained 47 different isolates by cultivation. They found ice nucleation activity in 14 isolates, of which 12 were bacteria and 2 were fungi. Eleven (9 bacteria, 2 fungi) of the 14 IN isolates were derived from precipitation samples and three isolates (bacteria) originated from the aerosol samples. The high fraction of different IN isolates, in particular from the precipitation samples, and number of species with so far unknown IN activity is surprising. Considering the overall limited cultivability of microorganisms under lab conditions, the results of this study indicate that microbial IN activity is far more distributed than previously assumed. The study is suited to the scope of the journal. The method is solid and the results are well presented. The manuscript is well written and well-structured and can be further improved as suggested below.
Specific comments:
Line 25: Better use INP as introduced in line 19 instead of “IN forming particles” as IN is not introduced here and based on IN definition in line 58 it would mean “ice nucleating forming particles”
Line 108: How were the filters pretreated for decontamination before aerosol sampling? Information on blank samples for aerosol sampling and handling should be added.
Line 124: Please add here also the aerosol samples. Moreover, I suggest to add the information given in line 140 about the volume (50 µL) and number of aliquots (30) already here as “microliter aliquots” covers a wide range of possible droplet sizes.
Line 155-157: Please add full information (or a reference) for the performed PCRs (PCR components, concentrations, cycling conditions). Note also, that ribosomal DNA in fungi is 18S and not 16S. Which primers were used for amplification of fungal 18S or did the bacterial primers only coamplify fungal 18S? This part needs some clarification on how the authors obtained fungal 18S sequences. The authors should also clarify and correct this in other parts of the manuscript., e.g., caption table S1, figure S11.
Line 360: As Cryptococcus and Metschikowia are not bacteria but fungi, please change caption to “Identities of 14 …IN bacteria and fungi”. Overall, both fungal species did not receive much attention in the manuscript although the title and abstract raised some expectations. The authors should add some discussion and comparison with the literature for the fungi they advertise in the title.
Line 424: Remove the “sp.” after “syringae” as syringae is the species name.
Line 441: please use “spp.” and not “sp.” if multiple species are meant.
Line 456: “Gammaproteobateria” – typo in bacteria, and missing hyphen (see line 345 Gamma-proteobacteria, be consistent).
Line 465: Lysinibacillus is not gram-negative. Please correct to gram-positive.
Line 490: Can the authors please add some more discussion and more specific suggestions on the “state-of-the art sequencing approaches” they mention here. I wonder how combining INP measurements with state-of the art sequencing should help to identify putative IN microbes that are not recovered by cultivation. The sequencing gives information about composition of the community, which are usually highly diverse, but only a small number of species possesses ice nucleation activity. A diversity analysis, however, does not give information about putative IN abilities of the organisms. Metagenomic (and transcriptomic approaches) are limited by database entries of IN genes, as these genes are not known for the many of the known IN organisms. Also note that some microorganisms (e.g., most known IN fungi) release cell-free IN into the environment. These IN would be covered by the IN measurements but as they do not contain DNA or RNA they would not be covered by the sequencing approaches. Furthermore, without cultivation it seems not feasible to proof the ice nucleation activity of a microorganism, even when (hopefully in future) gene similarities might suggest more candidates.
Table S1: Be consistent - genus and species names should be italics, “sp.” should not; contains several typos in e.g., Bacillaceae, Metschnikowiaceae. Paenibacillus is not a family but a genus, thus it should be Paenibacillaceae in the family column. Column blast identity has an extra comma in line Iso39, missing space in line iso3. IN ability column seems not needed, as IN onset temperature gives the “yes” or “no” information.
Table S4: Genus and species names should be italics, Iso5 – missing space, SSA18 – 7tewartia?
Figure S7: Typo in legend: Metschnikowiaceae; what does the line and the Y? at the right side of the legend mean?
Figure 4 and S10: It is confusing that the orange and yellow triangle symbols (sample 9) described in the legend point to a different direction in the plot. Caption for figure S10 needs to be checked “Sample numbers in the legend indication the precipitation”?
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AC1: 'Reply on RC1', Charlotte Beall, 10 Feb 2021
We thank the anonymous referee for their suggestions and thoughtful comments on how to supplement the discussion of the results. We include their comments and our responses below. Line numbers in our responses refer to the revised manuscript.
Specific comments:
Line 25: “Better use INP as introduced in line 19 instead of “IN forming particles” as IN is not introduced here and based on IN definition in line 58 it would mean “ice nucleating forming particles”
This has been corrected as suggested.
Line 108: “How were the filters pretreated for decontamination before aerosol sampling? Information on blank samples for aerosol sampling and handling should be added.”
Line 110 now reads: “Prior to sampling, filters were pretreated for decontamination by soaking in 10 % H2O2 for 10 minutes and rinsing 3X with ultrapure water.
Background levels of INPs from sampling handling processes were simulated using INP concentrations in aerosol sample field blanks assuming the average sampling volume (2270 L). Simulated INP concentrations across the 3 field blanks ranged between between 0 and 0.1 L-1 at -20 °C (see Fig. S6).”
Line 124: “Please add here also the aerosol samples. Moreover, I suggest to add the information given in line 140 about the volume (50 µL) and number of aliquots (30) already here as “microliter aliquots” covers a wide range of possible droplet sizes.”
Corrected as suggested. Line 129 now reads: “Briefly, the precipitation samples and aerosol sample suspensions were distributed in 24-30 50-microliter aliquots into a clean 96-well disposable polypropylene sample tray.”
Line 155-157: "Please add full information (or a reference) for the performed PCRs (PCR components, concentrations, cycling conditions). Note also, that ribosomal DNA in fungi is 18S and not 16S. Which primers were used for amplification of fungal 18S or did the bacterial primers only coamplify fungal 18S? This part needs some clarification on how the authors obtained fungal 18S sequences. The authors should also clarify and correct this in other parts of the manuscript., e.g., caption table S1, figure S11."
Thank you for bringing this to our attention. The 16S primers were able to capture the fungal 18S sequences and we did not use additional primers. We have added this clarification as well as the PCR reagents and cycling conditions to the manuscript.
Line 163: The PCR reaction contained 0.5 ng ml-1 genomic DNA, 0.2 mM of each primer, and 1x KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2601), and the thermocycler was set to the following program: 95°C for 30 seconds; 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; 72°C for 5 minutes.
Line 170: The 16S primers were able to capture 18S fungal sequences. Primers specific to 18S rRNA were not used.
Caption Table S1 and Fig. S11: 18S fungal sequences were obtained from 16S primers due to coamplification (see Methods Sec. 2.2).
Line 360: "As Cryptococcus and Metschikowia are not bacteria but fungi, please change caption to “Identities of 14 …IN bacteria and fungi”. Overall, both fungal species did not receive much attention in the manuscript although the title and abstract raised some expectations. The authors should add some discussion and comparison with the literature for the fungi they advertise in the title."
Corrected. Table 1’s caption now reads: Identities of 14 cultivable, halotolerant IN bacteria and fungi derived from aerosol or precipitation samples.
We agree that some discussion of fungal INPs is needed given the two fungal IN isolates featured here. Thank you for bringing this to our attention. The following paragraph has been added to Sec. 3.3, Line 391:
Fungal isolates Cryptococcus sp. and Metschikowia sp. represent two new ascomycotic and basidiomycotic IN fungal species, respectively, with INP concentrations 7-8 orders of magnitude lower than the highest reported values for fungal isolates F. armeniacum and F. acuminatum (Kunert et al., 2019). While other IN species of the Ascomycota and Basidiomycota phyla have been previously reported (e.g. Jayaweera and Flanagan, 1982; Kieft et al., 1988; Pouleur et al., 1992), very little is known regarding the distribution and source potential of fungal INPs. Moreover, multiple issues pose challenges to the differentiation of marine vs. terrestrial fungal species (Amend et al., 2019). Many fungi found in the sea are also found in terrestrial environments, and strong correlations with abiotic environmental conditions (Orsi et al., 2013; Tisthammer et al., 2016) and gene expression data (Amend et al., 2012) suggest that some fungi are truly amphibious. Issues with amplicon sequencing pose additional challenges due to coamplification of other eukaryotes and large biases toward terrestrial species in ITS rDNA primers, which were designed using sequence alignments from largely terrestrial representatives (Amend et al., 2019). However, future studies could take advantage of established marine fungi isolation and cultivation techniques to probe the INP source potential of various cultivable marine fungal species (e.g. Kjer et al., 2010; Overy et al., 2019).
Line 424: "Remove the “sp.” after “syringae” as syringae is the species name."
Corrected, thank you.
Line 441: "please use “spp.” and not “sp.” if multiple species are meant."
Corrected. Line 469 now reads: Additionally, whereas (Failor et al., 2017) reports high freezing temperatures between -4 and -12 °C for multiple Pseudomonas spp., none of the Pseudomonas spp. isolated in our study exhibited detectable IN activity.
Line 456: “Gammaproteobateria” – typo in bacteria, and missing hyphen (see line 345 Gamma-proteobacteria, be consistent).
Corrected, thank you.
Line 465: "Lysinibacillus is not gram-negative. Please correct to gram-positive."
Corrected.
Line 490: "Can the authors please add some more discussion and more specific suggestions on the “state-of-the art sequencing approaches” they mention here. I wonder how combining INP measurements with state-of the art sequencing should help to identify putative IN microbes that are not recovered by cultivation. The sequencing gives information about composition of the community, which are usually highly diverse, but only a small number of species possesses ice nucleation activity. A diversity analysis, however, does not give information about putative IN abilities of the organisms. Metagenomic (and transcriptomic approaches) are limited by database entries of IN genes, as these genes are not known for the many of the known IN organisms. Also note that some microorganisms (e.g., most known IN fungi) release cell-free IN into the environment. These IN would be covered by the IN measurements but as they do not contain DNA or RNA they would not be covered by the sequencing approaches. Furthermore, without cultivation it seems not feasible to proof the ice nucleation activity of a microorganism, even when (hopefully in future) gene similarities might suggest more candidates."
We agree that more discussion is needed here to explain how advanced sequencing approaches could help advance understanding of the factors that modulate bio-INP emissions. While it is indeed unlikely that we could identify a single IN species, advanced sequencing methods could illuminate relationships between specific communities and INP freezing activity. For example, high-throughput sequencing techniques for low biomass samples will enable sequencing of individual e.g. 50 μL droplets such that droplet assay measurements of INP concentrations could be related to communities present in low temperature vs high temperature freezing droplets (Minich et al., 2018). The referee also makes a good point about the inability of such methods to identify cell-free INPs. This paragraph has been edited as follows:
Finally, as cultivable populations represent a small fraction of the total microbial community, future studies should combine INP measurements with state-of-the-art sequencing approaches to identify relationships between specific microbial communities and INP freezing activity. Furthermore, a combination of advanced fractionation methods to identify the putative ice nucleating metabolites associated with specific microbial communities and computational networking could illuminate molecular and microbial linkages to ice nucleation and the mechanisms by which the entities work individually or in concert. Further study is also needed to understand the factors, such as atmospheric processing or nutrient limitation, that inhibit or enhance microbe IN behavior, as well as the factors that modulate the emissions of IN bacteria from the ocean surface.
Table S1: "Be consistent - genus and species names should be italics, “sp.” should not; contains several typos in e.g., Bacillaceae, Metschnikowiaceae. Paenibacillus is not a family but a genus, thus it should be Paenibacillaceae in the family column. Column blast identity has an extra comma in line Iso39, missing space in line iso3. IN ability column seems not needed, as IN onset temperature gives the “yes” or “no” information."
Thank you for pointing out the typos. These have been corrected. We agree that the column for IN ability is unnecessary and it has been removed.
Table S4: "Genus and species names should be italics, Iso5 – missing space, SSA18 – 7tewartia?"
Corrected.
Figure S7: "Typo in legend: Metschnikowiaceae; what does the line and the Y? at the right side of the legend mean?"
Corrected, thank you. The symbols are alpha and gamma (to indicate gamma vs alpha-proteobacteria).
Figure 4 and S10: "It is confusing that the orange and yellow triangle symbols (sample 9) described in the legend point to a different direction in the plot. Caption for figure S10 needs to be checked “Sample numbers in the legend indication the precipitation”?"
The triangle orientation has been corrected. (I also found another legend typo for Arthrobacter and Metschnikowia sp. typo and corrected).
The S10 caption typo has been corrected: “Sample numbers in the legend indicate the precipitation or aerosol sample from which the isolate was derived (see Table S3). Datapoints corresponding to isolates from aerosol are outlined in black.”
References:
Minich, J. J., Zhu, Q., Janssen, S., Hendrickson, R., Amir, A., Vetter, R., Hyde, J., Doty, M. M., Stillwell, K., Benardini, J., Kim, J. H., Allen, E. E., Venkateswaran, K. and Knight, R.: KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples, mSystems, 3(3), e00218-17, doi:10.1128/mSystems.00218-17, 2018.
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AC1: 'Reply on RC1', Charlotte Beall, 10 Feb 2021
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RC2: 'Comment on acp-2020-1229', Anonymous Referee #1, 05 Feb 2021
Terrestrial sources of microbial ice nucleators have been the subject of numerous recent investigations to understand their distribution in and meteorological impact to the atmosphere. Since studies to explore their potential marine sources are surprisingly fewer, this is a research are where more information is needed. This study analyzed aerosol and precipitation samples in attempts to enrich for marine microbes that displayed ice nucleation activity. Despite the biases inherent to this approach for, the authors were nevertheless successful in identifying a number of bacterial and fungal isolates that were tested and shown to be active as ice nucleators at temperatures warmer than -18C, and for one, near -2C. The microbiological and ice nucleating properties of the isolates, coupled with their probable sources in the air masses and precipitation samples analyzed, would be of interest to a broad community of researchers interested in marine and atmospheric science.
One general comment is that the results from fourteen isolates with IN activity are summarized in the abstract by presenting the temperature range of activities from -2.3 to -18C. I believe the truly interesting find in this work is the very warm IN temperature for the Brevibacterium strain, as I am not aware of another report of this phenotype in this phylum. My suggestion is to specifically emphasize this in the abstract as this will be a strain that will elicit interest from a range of microbiologists interested in novel mechanisms of biological ice nucleation.
Below are specific points the authors should consider when revising the manuscript:
Lines 32-33; 93-94; 330-332; 462: The phylogenetic information available cannot be used to definitively determine the environmental source of these isolates. The study observations collectively support that the principal aerosol source was marine, but the type and amount of sequence data obtained do not allow, for example, saying that Psychrobacter sp. 1b2 is marine and 2a is not. The phylogenetic resolution possible from the V4 region of the 16S/18S rRNA gene, which is about ~1/3 of the gene sequence, is useful for coarse phylogenetic assignments but is not able to resolve evolutionary relationships between closely related taxa. It is safe to conclude that the major source of cultured microbes was from aerosol samples with a marine origin and that many of the isolates are closely related to marine taxa but resolving environmental source from a few hundred nucleotides of small subunit rRNA sequences is not something that can be done with confidence.
Lines 110-111: Just to confirm that the aerosol samples used for culturing in media with ~35ppt salinity were initially placed in deionized water. It is important to note that this process would represent a significant osmotic shock to the cells. Also, assuming that a “hand” was not literally used for this aseptic procedure, so please clarify how the particles were removed from the filters.
Line 144: Just confirming if it was filtered and then autoclaved. Speculating it could be the reverse because a precipitate typically forms when autoclaving full strength seawater. Please also indicate the source of the seawater.
Line 155 and throughout manuscript: “16S V4 ribosomal DNA fragments were…” This is common lab slang that I suggest rewording here and throughout as "The V4 region of the 16S rRNA gene".
Lines 158-159: Suggest editing to something like “The sequences of the amplified 16S rRNA gene fragments were determined by...”
Lines 159 to 161: Please define acronyms on first use and describe the criteria used for OTU designation.
Line 163: Does this mean that the 16S rRNA gene sequences from different isolates were used to create some type of consensus sequence for each OTU? Please explain this process in more detail.
Lines 167-169: The description of this analysis is confusing to me. Distances >0.1 or 10% in the 16S rRNA gene would represent very large phylogenetic distances and not differences that would be confused at being the same "OTU". Please also indicate the length of DNA sequences used in this comparison.
Lines 206-209: It appears that the cells were washed and then tested immediately after nutrient removal. This would provide no opportunity for the microbes to respond to the experimental conditions, so it is difficult to interpret these results as being relevant to the effect of IN activity on the presence of nutrients.
Line 262: Is each period a separate rainstorm? There is more than one period for some days and with different trajectories in Figure 2, so maybe they are just different sampling periods.
Line 291: Please clarify if the sequence identities in Table S1 are BLAST outputs or based on distance matrices from multiple sequence alignments. Also, a general comment is that a >97% OTU estimate is highly conservative (e.g., Stackebrandt and Ebers 2006, Microbiology Today, 33:152-155).
Line 318-319: The isolated bacterial and fungal taxa cannot easily be compared to microbial communities, which are associations of many many different types of interacting microbes. And if they are Pacific-sourced aerosols, I’m not sure to consider it a "a warmer climate" even though that is the case where they were deposited in SoCal. Can the isolates grow at cold temperatures or is there any other evidence for cold tolerance, if that is in fact what is being implied here? Please revise this sentence for clarity.
Line 334: Is it known if the isolates have optimal growth at salt concentrations in seawater, not just tolerance to the concentrations in the seawater-based media? That would provide physiological support for a marine lifestyle.
Lines 384-385: Does this means than isolates having identical sequences in the portion of the 16S rRNA gene examined were deemed clonal and that one isolate was selected as a representative?
Lines 388-390: Were any of the observations in Figure S11 replicated to confirm that the patterns of IN at these temperatures and isolates were not more affected by the age of cultures or other potential variations in the way the cultures were handled between experiments?
Lines 411-420; 468: Ample time after removal of nutrients may not have been provided in these experiments and the authors should consider limiting this discussion. The one conclusion that can be made is that the activity observed does appear to be associated with the cells and not removed by washing, suggesting the nucleating material is membrane bound or associated with the cell envelope.
Lines 429-432: Please note that the percentage of cells that serve as INPs is temperature dependent, and the "active" fraction values cited from the literature are likely referring to very warm subzero temperatures, whereas for P. syringae populations at temps below -10C, values approaching 100% could be expected. This leads me to suspect the caveats stated are valid for INPs that activate in the -2C range (i.e., Brevibacterium) but may be overly conservative for the colder temperatures of ice nucleation observed in their experiments.
Lines 436-440: The connection being made with the Failor et al. study is ambiguous. Please clarify if the suggestion is that the taxa in the Failor study were of a marine source, that in the isolates in this study were not marine in origin, or something else entirely.
Line 449: I am not able to find where this is discussed further. This suggests some of the IN activities reported were difficult to repeat, which would be consistent with other similar attempts, Failor et al. being one good example.
Line 484-486: General comment to authors: I am most surprised by the fact that out of a group of less than 50, you found one that is active at warmer than -5C and is a member of a phylum where I am not aware of other known examples of this phenotype. In my view, this single isolate may be the most important contribution of this study and will be of interest for additional work to decipher if the mechanism of ice nucleation differs from that of certain Gammaproteobacteria.
Data availability: Please provide database accession information to access the DNA sequence data from this study.
Figure 3: Please indicate the number of aligned nucleotides and method of phylogenetic analysis used for evolutionary tree construction.
Charlotte M. Beall et al.
Data sets
Data from: Cultivable, halotolerant ice nucleating bacteria and fungi in coastal precipitation Charlotte M. Beall, Jennifer M. Michaud, Meredith A. Fish, Julie Dinasquet, Gavin C. Cornwell, M. Dale Stokes, Michael D. Burkart, Thomas C. Hill, Paul J. DeMott, and Kimberly A. Prather https://doi.org/10.6075/J0GQ6W2Z
Charlotte M. Beall et al.
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