PONE-D-19-20101

Isolation and Diversity of Sediment Bacteria in the Hypersaline Aiding Lake, China

PLOS ONE

Dear Dr Guan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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The full referee’s comments are indicated at the end of this letter.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes the isolation of bacteria from sediments collected from a hypersaline lake. A diversity analysis of these bacterial isolates based on the evolutionary relatedness of the 16S rRNA gene was also performed. Overall, the study presents new and interesting data with regards to the potential to access novel species and genera via a culture-based approach and highlights the value of exploring environments that are unique and underexplored. Here are some comments and recommendations for the authors:

1. Abstract: It would be helpful to the reader to understand the aim of the study at the outset and therefore should be included in the Abstract.

2. Line 30: Insert the word ‘supplemented’ before ‘with 5% of 15% (w/v) NaCl’

3. Line 31: The use of the word ‘significantly’ implies statistical analyses – if any were performed, please provide details in the methodology section and provide the p-value.

4. Line 37: Values below 20 is typically written out in full, therefore it should be ‘…two novel genera…’ and ‘…four novel species…’

5. Line 38: Since reference is made to more than one genus, the plural, ‘genera’, should be used.

6. Line 53: The sentence is ambiguous, implying that the novel species described are different forms of culture-dependent methods. To avoid the ambiguity, it is recommended that the sentence should be changed to: ‘…have been described using culture-dependent methods: Brevibacterium salitolerans…’

7. Line 59: It is mentioned that the Turpan Basin is the hottest region in China – can you provide a temperature range to provide insight into the environment from which the samples were collected?

8. Line 60: Please provide a reference to support the statements regarding temperature and salinity.

9. Line 62: ‘bacteria diversity’ should be ‘bacterial diversity’

10. Site description and sample collection: Are three sediment samples sufficient to serve as representative samples of the whole lake area? If they were not meant to be representative samples, then I would suggest changing the manuscript title to reflect this.

11. Line 77: Sediments will not dissolve in distilled water – rather state that the sediments ‘were resuspended in distilled water’.

12. Line 86: What was the rationale behind the design of the different types of isolation media? Even though emphasis is made that these different types of media allowed for access to unique bacterial isolates, the significance of this is not really discussed or elaborated on in the manuscript. The section on this in the Discussion should therefore be expanded on. In addition, why was the particular pre-treatment method employed in this study? Do the authors feel that this also contributed towards the isolation of a wider variety of bacterial strain?

13. Lines 87-88: Stating that ‘The compositions of the nine media are shown in Table 1’ is redundant and should be deleted. Reference to the media have already been made in line 86.

14. Line 90: Is there any particular reason why an incubation temperature of 37�C was used? It is 12-14 degrees higher than the temperature recorded for the areas where the sediment samples were collected.

15. Line 111: The use of a 99% sequence identity based on 16S rRNA gene sequences as the basis for clustering into one operational taxonomic unit poses great risk of clustering isolates that are novel. It is often found in actinobacterial genera in particular where unique species share a high 16S rRNA sequence similarity, sometimes even 100%. An alternative method of dereplication should’ve been employed – either a sequence-based method (use of another taxonomic marker) or culture-based (phenotypic differences).

16. Lines 119-125: Does the Geology of the region support the differences in the physicochemical properties of the three sediment samples? Is there any correlation between the isolates (genus/family) and the sediment sample isolated from?

17. Lines 145-148: Make sure that all mention of ‘strains’ are correct – in some instances, the singular, ‘strain’ is used instead of the plural form.

18. Lines 155-156: Correct the sentence structure – ‘…suggesting that these may represent two novel genera of Bacillaceae’.

19. Line 170: ’17 families’ not ’17 family’.

20. Line 171-172: A statement is made here that most bacterial groups were isolated on one specific medium – this needs to be expanded on in the discussion section.

21. Line 184: ‘…one novel genera’ should read ‘…one novel genus’.

22. Line 195: ‘…16S rDNA…’ should be ‘…16S rRNA genes…’

23. In the discussion section, mention is made of the various genera isolated from other types of salt lakes. However, different media and isolation techniques would’ve been used to that of the study reported here. Comparisons can therefore not be made among these studies. Did any of these studies report on community analysis?

24. Lines 215-216: ‘Micromonospora’ is mentioned twice.

25. Lines 229-230: See comment above drawing comparisons.

26. The aspect that should be highlighted in the discussion section, is the value of the different media types in accessing different genera/species. Are there any reasons why certain genera were isolated on specific media? Any correlation to the physicochemical properties of the sediment samples?

27. Please make sure that the manuscript is edited for grammar and language usage. Some examples are listed above, but this is not a comprehensive evaluation.

Reviewer #2: The manuscript (MS) by Guan and colleagues describes the isolation of novel bacteria from a hypersaline lake in China. The MS is interesting but IMHO contains several flaws that require attention before it can be accepted for publication as I point below.

General comments.

1. During the MS when referring to the name and/or lists of different genera, the authours sometimes use an alphabetical order and sometimes they don’t. They should be more homogenous through the entire MS. For instance, the abstract is not in alphabetical order but in lines 172 to 174 the names of the genera mentioned are precisely in alphabetical oder. It is nt critical to use one or another but homogeneity would definitively be preferred.

2. It should be clear for the authors that not every reader ifs familiar with the geography of China. Hence, I would stongly suggest that for the final versión of the MS, the authours include a map of China showing the exact location of the lake. This can be referred in section 2.1 Site descriptions and sample collection. In addition, is there any reference for the sentence “The high salinity, low nutrients level, dry climate, and high UV intensity makes it an extreme environment.” (Lines 69-70). If there is any, then it should be added to the MS.

3. Section 2.2. There is a bunch an wide range of seletive isolation media and the authors claim that they used 9 (line 86, Table 1). However when checking Table 1, all but one (ie. eight) are media “From this study”. This is intriguing because when other academics might check the MS in it final form they can argue why and/or how these eight media were chosen since this is not included nor mentioned anywhere in the current form of the MS. Therefore, I believe that the authours should add a line and/or sentence and/or paragraph indicating why and how these media was chosen for the study.

4. It is not clear to me why there are only 70 sequences deposited in GenBank (MK818765-MK818834 = 69 sequences plus MK296404 = 70) if the authors mentioned that they isolated 343 bacteria. That is only a mere 20%. If the identifiction of the bacteria is purely based on 16S rDNA gene sequencing then I think they should, at least, submit 50-60% of their sequences to GenBank and not saying that there is diversity but not providing the corresponding sequences for their isolates.

5. Section 3.1. According to the results from the authors, they sampled in three different places and provide the information in table 2. In the MS they also indicate that the conditions of each sample are different and this is very well exemplified by the high bacterial diversity. Since the authors employed 9 different media in the study, then I would strongly suggest a table indicating the number of isolates recovered from each sediment. Perhaps there is one sediment that showed a higher degree of diversity over the others. These would also help the authors to decide how many other sequences can be submitted to GenBank rather than submitting all of them as I suggested for point number 4.

6. Also, how did the authors checked for “duplicating strains”? Again, is this only base don the 16S rDNA gene sequencing of the isolates? This is indicted in Section 3.2, lines 127-128.

7. I am not convinced by the fact that a 97% similarity (less) refers to a different species. There are examples of either a novel species or a putative novel genera. This should be handle with care. If they haven’t done so, then I suggest that the authors also genome sequence some of the most interesting isolates (ie the ones that are the most different ones acoording to EZBiocloud) and then make or at least mention DNA-DNA in silico (available from the DSMZ website) to highlight this point. However I would strongly object adding the full genome sequencing information to this MS or it would definitively become a never ending story.

8. Section 3.2 lines 143 – 148. If the reader is not familiar with families and genera, these lines become extremely confusing because there is n indication of which genera is either Actinobacteria, Firmicutes, Proteobacteria or Rhodothermaeota. This is definitively a very interesting paragraph because it certainly highlights the diversity found in the study but there is no separation between any phyla thus making it difficult and confusing.

Due to this number of general comments, then I find the Discussion section hard to follow. Besides, the English should definitively be improved in order to exploit the full potential of this MS as there are not too many studies on extreme lake environments.

I should mention that I find the MS very interesting but I am afraid in its present form I would be against its publication mostly because of its current form. It can certainly hav a higher impact if it is re-arranged/presented in a better way and hopefully the authors would be willing to incorporate all of the comments.

Minor comments.

1. Section 2.3, line 97. I believe reference 25 must be the one for the EZBiocloud website. Apparently some references are misplaced causing confusion because also reference 26 is not the one for the sentence.

2. Section 2.3, line 102. How many base pairs were obtained for the 16S rDNA gene sequence? This is not indicated or are the authors assuming that anyone will check the size of the sequences deposited in the GenBank database?

3. Line 231, shouldn’t it be “are novel” instead of “as novel”?

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Reviewer #1: No

Reviewer #2: No

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Isolation and Diversity of Sediment Bacteria in the Hypersaline Aiding Lake, China

PONE-D-19-20101R1

Dear Dr. Guan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No