Culturable Microorganisms of Aerosols Sampled during Aircraft Sounding of the Atmosphere over the Russian Arctic Seas

Round 1

Reviewer 1 Report

I find the article titled "CULTURABLE MICROORGANISMS OF AEROSOLS SAMPLED DURING AIRCRAFT SOUNDING OF THE ATMOSPHERE OVER THE RUSSIAN ARCTIC SEAS" (atmosphere-2888529) to be interesting. The purpose of this study was to obtain data on the vertical distribution of bacteria and fungi in atmospheric aerosols during aircraft sounding of the atmosphere over the seas of the Arctic Ocean of the Russian sector. However, there are major  corrections that require attention before considering the article for publication:

1.    Abstract Section: "The data obtained indicate the need for systematic study of atmospheric microbiota composition to prevent population-related diseases." I believe that the microbiota of the external environment cannot be controlled. I suggest replacing the word "control" with "study" or another suitable term. Please revise throughout the manuscript, as the same error is repeated in the introduction and discussion sections.

2.    Graphical abstract:  consider adding a graphical abstract.

3.    Materials and methods section: Figure 1 should be considerably improved so that flight route can be better viewed.

4.    Materials and methods section: Table 1 should be placed in section 2.2. not in section 2.3 as it is at present.

5.    Materials and methods section. 2.3. The concentration of culturable microorganisms. ``lower fungi``?. Use current fungal terminology throughout the manuscript.

6.    Materials and methods section. 2.7. Microorganisms’ antibiotic properties. ``The antibiotic activity of spore-forming bacteria was studied by the method of delayed antagonism [36] against opportunistic test strains: Escherichia coli 6645 ATCC 25922, Candida albicans 620 Y-583, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae B-378 B-4894 and Bacillus cereus ATCC 1070 2 from the collection of bacteria...´´. Should improve the wording, since Candida is a yeast, not a bacterium.

7.    Materials and methods section. 2.8. Microorganisms’ taxonomy. In fungi taxonomy because ITS 5.8s was not used?. did they use 16 s for fungi?

 I advise the authors to see the following articles: Schoch, C. L. et al. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl Acad. Sci. USA 109, 6241–6246. https://doi.org/10.1073/pnas.1117018109.

White, T., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfand, D., Shinsky, J., White, T. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, Innis, M, pp. 315–322.

8.    Result section: Micromycetes?. Is a mycological term in disuse. It does not refer to genera such as Penicillium, Aspergillus, and Aureobasidium.  Use current fungal terminology throughout the manuscript.

9.    Result section. 3.2. Antibiotic activities of isolates. "S. epidermidis" should be in italics as this is the format for writing scientific names. Please review the entire manuscript to ensure this error is corrected and not repeated.

10. Result section. Figure 3.  Керосин?. Please provide the English translation and check the English spelling throughout the manuscript.

11. Discussion section. In the final part include the limitations of the work (you can focus on your sampling method and the equipment used). Future research directions may also be mentioned.

12. Conclusion section. The conclusions section should be completely rewritten. In its present form it is a summary of results and is too long. Provide conclusions according to the objective of your manuscript.

13. Supplementary Materials section. I find it a bit excessive to have 20 figures showing the backward trajectory of the air masses. Show only the most relevant locations, such as the areas of highest microbial concentration. Moreover, you should provide a table with the meaning of each acronym from Sp1 to Sp146.

Minor editing of English language required

Author Response

1. Abstract Section: "The data obtained indicate the need for systematic study of atmospheric microbiota composition to prevent population-related diseases." I believe that the microbiota of the external environment cannot be controlled. I suggest replacing the word "control" with "study" or another suitable term. Please revise throughout the manuscript, as the same error is repeated in the introduction and discussion sections.

We changed “Obtained data lead to the necessity of systematic control of atmospheric microbiota composition to prevent population diseases” to “Obtained data lead to the necessity of systematic study of atmospheric microbiota composition to be ready to combat with emerging population diseases”. The similar corrections are made throughout the text, se blue font in the text.

2. Graphical abstract: consider adding a graphical abstract.

We added Graphical abstract.

3. Materials and methods section: Figure 1 should be considerably improved so that flight route can be better viewed.

We reconstructed Figure 1, see text version V1.

4. Materials and methods section: Table 1 should be placed in section 2.2. not in section 2.3 as it is at present.

Table one is moved to section 2.2.

5. Materials and methods section. 2.3. The concentration of culturable microorganisms. ``lower fungi``?. Use current fungal terminology throughout the manuscript.

We changed “for identifying lower fungi and yeast” to “for identifying fungi” as soon as yeasts are fungi too.

6. Materials and methods section. 2.7. Microorganisms’ antibiotic properties. ``The antibiotic activity of spore-forming bacteria was studied by the method of delayed antagonism [36] against opportunistic test strains:

Escherichia coli 6645 ATCC25922,

Candida albicans 620 Y-583,

Staphylococcus aureus ATCC 6538,

Klebsiella pneumoniae B-378 B-4894 and

Bacillus cereus ATCC 1070 2 from the collection of bacteria...´´. Should improve the wording, since Candida is a yeast, not a bacterium.

We improved the wording: “The antibiotic activity of spore-forming bacteria was studied by the method of delayed antagonism [36] against opportunistic test strains of bacteria and yeast:…. “

7. Materials and methods section. 2.8. Microorganisms’ taxonomy. In fungi taxonomy because ITS 5.8s was not used? .did they use 16 s for fungi?

I advise the authors to see the following articles:

Schoch, C. L.et al. 2012. Nuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for Fungi. Proc. Natl Acad. Sci. USA 109, 6241–6246.https://doi.org/10.1073/pnas.1117018109.

White, T., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfand, D., Shinsky, J., White, T. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, Innis, M, pp. 315–322.

We added information as follow (see text in blue font also):

Fungi were identified by morpho-physiological characteristics, and molecular genetic methods were used selectively for small number of strains.

ITS (intergenic ribosomal spacer sequence) was used for identification of fungal strains by molecular genetic methods. The amplification reaction of ITS-fragment of genomic DNA was carried out using oligonucleotides ITS1 and ITS4 for the first round of PCR (obtained fragment ~ 700 bp), ITS3 and ITS4 for the second round of PCR (300 bp). Sequencing reaction of the obtained PCR fragments was performed using the same oligonucleotides and BigDye v.3.1 reagent (Applied Biosystems, USA) under standard conditions. The obtained sequences were analyzed using ABI Sequence Scanner and FinchTV 4.1 software. Sequences were compared with those available in the NCBI GenBank database (ncbi.nlm.nih.gov) using the BLASTN algorithm, and phylogenetic analysis was performed using the MEGA 7.0.21 program.

8. Result section: Micromycetes?. Is a mycological term in disuse. It does not refer to genera such as Penicillium, Aspergillus, and Aureobasidium. Use current fungal terminology throughout the manuscript.

Corrected (see text in blue, line 225)

9. Result section. 3.2. Antibiotic activities of isolates. "S. epidermidis" should be in italics as this is the format for writing scientific names. Please review the entire manuscript to ensure this error is corrected and not repeated.

Corrected (see text in blue, line 313)

10. Result section. Figure 3. Керосин?. Please provide the English translation and check the English spelling throughout the manuscript.

Corrected

11. Discussion section. In the final part include the limitations of the work (you can focus on your sampling method and the equipment used). Future research directions may also be mentioned.

Global climatic changes and increasing anthropogenic load imply further changes in the Arctic biota, enrichment of the air environment with microorganisms that were not typical before. The results obtained are preliminary and need further research. For an objective assessment of the data obtained, additional flights with route repetition are required. Further studies of the Arctic microbiota are planned as part of a comprehensive expedition using the Tu-134 Optik laboratory aircraft. Unfortunately, aircraft sounding is an expensive research method and cannot be used as much as researchers would like in order to obtain the most correct data. We hope that integration of international research programs will help to obtain comprehensive monitoring results.

12. Conclusion section. The conclusions section should be completely rewritten. In its present form it is a summary of results and is too long. Provide conclusions according to the objective of your manuscript.

We change conclusion to text below:

Analysis of the studied samples of Arctic atmospheric aerosols revealed a significant diversity of culturable bacteria and fungi with concentrations averaging 5.5 × 10³ CFU/m³, regardless of altitude and sampling location at altitudes from 200 m to 10,000 m. 

The presence in aerosols of opportunistic fungi and bacteria, including representatives of the genus Staphylococcus and antibiotic-resistant bacteria, makes it necessary to control the microbiological composition of aerosols to prevent infectious diseases.

The discovered highly effective psychrotolerant bacteria - oil destructors are suitable for the creation of consortia of microorganisms, for use in the remediation of oil-contaminated areas of Siberia and the Arctic.

13. Supplementary Materials section. I find it a bit excessive to have 20 figures showing the backward trajectory of the airmasses. Show only the most relevant locations, such as the areas of highest microbial concentration.

20 figures showing the backward trajectory of the airmasses ware restricted to 6 figures for the locations of highest microbial concentration. See Supplementary Materials V1. And we added “Only some backward trajectories for samples with highest concentrations of culturable microorganisms went from more southern regions”.

Moreover, you should provide a table with the meaning of each acronym from Sp1 to Sp146.

Microorganism isolates are presented in order of detection from Sp1 to Sp146 simply. Detailed description of these strains is too big to be included into the text and even into Supplementary Materials.

• Comments on the Quality of English Language
• Minor editing of English language required

Author Response File: Author Response.docx

Reviewer 2 Report

This paper presents the analysis of microorganic concentrations, collected in 24 samples in the troposphere over Russian Arctic during several flights in September 2020. The paper is very interesting, well written, and can be published if the following minor comments are considered.

Please provide the line numbers, to make the review process easier.

End of part 2.3: What the authors mean by ±0.2 log10 ? Of which value they are speaking?

Part 2.6 consists of only one sentence that is too long.

Jus before Figure 2: I don’t understand why there is the sign “-“ before the percentage, as well as before “175”. Also, later in the text.

7 lines above table 2: Can the authors explain what “gram-negative” means?

Author Response

Comments and Suggestions for Authors

This paper presents the analysis of microorganic concentrations, collected in 24 samples in the troposphere over Russian Arctic during several flights in September 2020. The paper is very interesting, well written, and can be published if the following minor comments are considered.

Please provide the line numbers, to make the review process easier.

Done. Please see V1 text’s version

End of part 2.3: What the authors mean by ±0.2 log10 ? Of which value they are speaking?

Usually, the number of microorganisms is presented in logarithmic scale. So ±0.2 log₁₀ is confidential interval for average value of microorganisms’ number.

Part 2.6 consists of only one sentence that is too long.

We rearrange part 2.6 into3 sentences:

Microbial cultures isolated from Arctic aerosols were tested for their ability to degrade oil by sowing on agarized and liquid 8E medium [34].  Up to 1-5% of high-viscosity oil from the Usinsk field (Komi Republic, Russian Federation) was added to the medium as the only carbon source.  The average density of oil was 0.920-0.986 g/cm³.

Jus before Figure 2: I don’t understand why there is the sign “-“ before the percentage, as well as before “175”. Also, later in the text.

We remove signs “-“ in this part of the text.

7 lines above table 2: Can the authors explain what “gram-negative” means?

It’s a misprint: there should be  Gram-negative.

"Gram-negative", «Gram-positive» - culture cells that stain negatively by Gram's method.

Gram-negative and Gram-positive bacteria is the accepted terminology for bacteria whose cells stain negatively or positively by Gram's method. The difference in cell staining is based on differences in cell wall structure.

Round 2

Reviewer 1 Report

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