Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanites (Slovakia)
Round 1
Reviewer 1 Report
The article ,,Isolation and Identification of Bacterial Communities in Neutral Mine Drainage in Central Slovakian Neovolcanitoes (Slovakia)‘‘ is interesting and valuable. I have a few comments:
- The introduction is too broad, I would recommend shortening it;
- Old literature is cited. The bibliography contains only 10 articles published in 2018 and later. I recommend supplementing the discussion with data from new articles;
- The first 4 conclusions are not specific and not supported by the research results. I recommend removing them.
Author Response
Dear Reviewer,
Thank you for your comprehensive and detailed review report.
I believe that the manuscript will be at a significantly higher level after incorporating the comments, and will be recommended for publication.
All your specific comments are included in the edited text of the manuscript in the attachment, visible via track changes.
Please see the attachment
Point 1: Does the introduction provide sufficient background and include all relevant references? The introduction is too broad, I would recommend shortening it
Response 1: The introduction was shortening, and prepared so that sufficient background provided and relevant references were including.
Point 2: Old literature is cited. The bibliography contains only 10 articles published in 2018 and later. I recommend supplementing the discussion with data from new articles.
Response 2: The discussion has been supplemented with regard to the current relevant literature sources, that has been added to the references also.
Point 3: The first 4 conclusions are not specific and not supported by the research results. I recommend removing them.
Response 3: The conclusions was updated taking into account research results. Please see the attachment
Reviewer 2 Report
The manuscript by Prepilkova et al. submitted to Water (ID: 2141967) characterizes bacterial population of mine drainage in the area of Central Slovakian neovolcanitoes. The authors have used different methods to examine the bacterial community. Investigations pertaining to the above is relatively fewer in mines and the results obtained would improve our knowledge on the bacteria that inhabit in such environments. Why did the authors choose cell culture/isolates to characterize the bacterial population in the mine water? The authors would be aware that most of the bacteria are not cultivable on agar plates and definitely miss those communities which are not cultivable. In the approach followed by the authors, lot of bias could be involved while choosing the isolates in deciphering the bacterial community from mine waters. Instead the authors should directly have extracted the bacterial DNA from the mine water and subjected to 16Sr RNA gene analysis, which could have given a broader community profile. For identification, contrasting results were obtained between the
16S rRNA gene sequence and MALDI-TOF mass spectrometry which authors themselves point out. This disparity could have been removed by simply following the first approach. Such could lead to confusion. Overall writing needs to be improved. Too much of description in the introduction and discussion section.
Specific comments
L14-15: sentence incomplete
L17-19: identification appears twice in the sentence.
L19-20: approximately equally? Please rewrite it.
L22: % should be in brackets
L22-34: define what role can these bacteria play in such environments
L33: almost and always are not the same. Please rewrite the sentence
Introduction: Too much of description of study site and related information. Hypothesis, aim and objectives are missing. Need more to define the role of bacteria in such environments
Table 1: for Hodrusa district, why two pairs of quotation marks?
L179-184, L197-200: bias could be involved in choosing the isolate to represent bacterial community in mine waters.
L205-211: differences identification between two approaches
Discussion: avoid repetition of results. Define what ecological role do the identified bacterial communities play in such environments. Eg. Bioremediation, cycling of elements etc.
Author Response
Dear Reviewer,
Thank you for your comprehensive and detailed review report.
I believe that the manuscript will be at a significantly higher level after incorporating the comments, and will be recommended for publication.
All your specific comments are included in the edited text of the manuscript in the attachment, visible via track changes. Please see the attachment
Point 1: Why did the authors choose cell culture/isolates to characterize the bacterial population in the mine water? The authors would be aware that most of the bacteria are not cultivable on agar plates and definitely miss those communities which are not cultivable. In the approach followed by the authors, lot of bias could be involved while choosing the isolates in deciphering the bacterial community from mine waters.
Response 1: We wanted to test the isolates for the possibilities of use in biotechnology. We can do this only with those microorganisms that we can obtain after cultivation, because we consequently tested their growth on agar with different concentrations of heavy metals, biosorption from the medium also. The results we would published in the next article after ending of all experiments. In short, we wanted to further test the bacteria, not only do "screening". I think this answers most of the questions and explains our goal.
Point 2: Overall writing needs to be improved. Too much of description in the introduction and discussion section.
Response 2: The introduction was shortening, the discussion has been carefully revised.
Point 3: Introduction: Too much of description of study site and related information.
Response 3: The introduction was shortening, and prepared so that sufficient background provided and relevant references were including.
Point 4: Hypothesis, aim and objectives are missing.
Response 4: The aim of the research was to isolate bacteria from contaminated environments suitable for use in biotechnology. In the attached revised manuscript, the conclusions summarize that this is the first information on the microbiological composition for the of the researched area and that the bioremediation potential of these isolates should be further studied.
Point 5: Comments lines
L14-15: sentence incomplete
L17-19: identification appears twice in the sentence.
L19-20: approximately equally? Please rewrite it.
L22: % should be in brackets
L33: almost and always are not the same. Please rewrite the sentence
Table 1: for Hodrusa district, why two pairs of quotation marks?
Response 5: All your specific comments are included in the edited text of the manuscript in the attachment, visible via track changes.
Point 6: Comment lines L179-184, L197-200: bias could be involved in choosing the isolate to represent bacterial community in mine waters.
Response 6: The authors agree with this opinion, but we did not have the opportunity to use such extensive research of environmental bacterial DNA from the mine water yet, we will work on it to improve the determination of bacterial communities in the environment in next coming study.
Point 7: Comment lines L205-211: differences identification between two approaches
Response 7: From fresh culture (24-h) of each isolate, analysed by direct transfer (ICP) MALDI TOF method, measured spectra did not yield a satisfactory match. Most of isolates ended up being unreliable identified. For these cases, the extraction method of MALDI TOF determination is recommended.
Point 8: Comment line L22-34: define what role can these bacteria play in such environments
Response 8: Our data confirm the presence of bacterial species occurring in environment polluted by potentially hazardous elements, what may indicate their bioremediation potential.
Point 9: Discussion: avoid repetition of results. Define what ecological role do the identified bacterial communities play in such environments. Eg. Bioremediation, cycling of elements etc.
Response 9: This comments are included in the edited text of the manuscript in the attachment, visible via track changes. Please see the attachment
Reviewer 3 Report
Drainage waters with a diverse elemental composition and pH are a special habitat for various microorganisms. The study of their physiological and biochemical properties makes it possibility the adaptive capabilities of certain species and to identify useful ones for biotechnological purposes.
The proposed article differs from the traditional presentation scheme, which makes it difficult to understand the results of the study and discuss them. The Introduction describes the mine waters of Central Slovakia and does not discuss the diversity of microorganisms in similar ecosystems, their composition and physiological characteristics. Partially, these data are presented in the discussion, which does not exclude the citation of similar data in the introduction. The purpose of the research and the tasks are not discussing. The title of the paper needs to be clarified, only cultivated bacteria are isolated and identified, and not the all bacterial communities. Drainage water chemistry data are best presented in Table and discussed in the Materials and Methods. This will allow you to evaluate the difference between the studied samples and compare with others.
2.1. Sampling and storage
How were the samples taken, close to the shore or using a boat? Did you measure the pH of the water at the same time? How many samples were taken for analysis in one area?
117, 121 - Nutrient Agar - check spelling on different lines.
125 -Olympus BX (manufacturer country?)
153- missing Supplementary file
177 - Do you registered the received sequences, and checked for chimeras?
Table 3 requires revision. The title is - Comparison of identification: results by MALDI-TOF mass spectrometry, 16sRNA sequencing and BLAST analysis. The table shows the Gram staining and BLAST values. 16sRNA sequencing = BLAST analysis? Discuss the percentage of similarity.
213-219 Which species are gram-negative, this is not in the table.
220 What data are reported obtained using by MALDI-TOF mass spectrometry or 16S rRNA sequencing?
In the Discussion, could provide a table with a list of core taxa living in drainage waters with different pH and compare this data with other regions of Slovenia and in the world. Since the authors obtained a small amount of data about of the diversity of cultivated strains, the assessment of the diversity of microbial communities is not entirely correct. It would be more interesting to study the physiological and biochemical parameters of the isolated strains and their ability to accumulate or precipitate various elements that are present in drainage waters. Estimate the concentration of metals is preferring by certain species and estimate the pH affect their vital activity.
Author Response
Dear Reviewer,
Thank you for your comprehensive and detailed review report.
I believe that the manuscript will be at a significantly higher level after incorporating the comments, and will be recommended for publication.
All your specific comments are included in the edited text of the manuscript in the attachment, visible via track changes.
Please see the attachment
Point 1: Sampling and storage: How were the samples taken, close to the shore or using a boat? Did you measure the pH of the water at the same time? How many samples were taken for analysis in one area?
Response 1: For microbiological analysis we taken one water samples per site, sampling was done close to the shore. On-site pH measure were done using inoLab®Multi 9310 IDS (WTW Weilheim Xylem Analytics Germany) (measured values in Table 1.).
Point 2:
Comment lines 117, 121 - Nutrient Agar - check spelling on different lines
Comment line 125 -Olympus BX (manufacturer country?)
Response 2: All your specific comments are included in the edited text of the manuscript in the attachment, visible via track changes.
Point 3: Comment line 153- missing Supplementary file
Response 3: Supplementary file is filled out with measurement data, please see the attachment.
Point 4: Comment line 177 - Do you registered the received sequences, and checked for chimeras?
Response 4: We did not register the received sequences.
During our first sequencing procedures, we observed large primer-dimer peaks, we discussed the errors with the SEQme sequencing service*, after that we proceeded to new workflows, developed new sequences, where we obtained reliable results without chimeras.
*In some samples (e.g. isolate 4.1.), there are large primer-dimer peaks at the beginning of the sequencing read (high fluorescence signal), clearly visible in the raw data, after which the normal sequence continues. As part of the data analysis, we eliminated these peaks from the electropherogram, as it is a mixed/unreadable sequence.
Point 5: Table 3 requires revision. The title is - Comparison of identification: results by MALDI-TOF mass spectrometry, 16sRNA sequencing and BLAST analysis. The table shows the Gram staining and BLAST values. 16sRNA sequencing = BLAST analysis? Discuss the percentage of similarity.
Response 5: All your specific comments are included in the edited text of the manuscript in the attachment, visible via track changes.
The percentage of similarity. The sequencing data for each isolate was compared with sequences producing significant alignments and choose via highest percentage identity.
Point 6: Comment lines 213-219 Which species are gram-negative, this is not in the table.
Response 6: Table 3. is corrected with accordance to your comment and provide the results by Gram staining, MALDI TOF mass spectrometry and BLAST analysis of 16s rRNA sequences.
Point 7: Comment line 220 What data are reported obtained using by MALDI-TOF mass spectrometry or 16S rRNA sequencing?
Response 7: Both, the results are discussed relevant to the method of analysis. In this line is commented the isolate 1.5. and its identification by Gram staining and MALDI-TOF mass spectrometry.
Point 8: In the Discussion, could provide a table with a list of core taxa living in drainage waters with different pH and compare this data with other regions of Slovenia and in the world
Response 8: The diversity of bacterial colonization of the mine waters in the localities of Slovakia is described in detail in the discussion.
This comment is included in the edited text of the manuscript added in the attachment, in the form Table 4. "Comparison of microbial communities in mine drainage on different sites" and incorporated into the discussion, visible via track changes.
Point 9: Since the authors obtained a small amount of data about of the diversity of cultivated strains, the assessment of the diversity of microbial communities is not entirely correct. It would be more interesting to study the physiological and biochemical parameters of the isolated strains and their ability to accumulate or precipitate various elements that are present in drainage waters.
Response 9: We state that, we want to continue working with those isolates, and after the end of the experiments we will prepare next article about our results. We did it, nowadays we continue the experiment, the results are quite extensive so far (many concentrations of many elements, even biosorption).
Point 10: Estimate the concentration of metals is preferring by certain species and estimate the pH affect their vital activity.
Response 10: We don't know exactly what about Reviewer thinking in this comment.
Should we changed the conditions for those isolates or measured the concentrations of heavy metals in the samples?
If Reviewer wants to know concentrations, we have them, and we can supplement it, but testing the resistance of isolates and reaction of communities to changed conditions will be completed later, in another article that we are working on.
Point 11: The title of the paper needs to be clarified, only cultivated bacteria are isolated and identified, and not the all bacterial communities.
Response 11: The authors agree with this opinion, but we did not have the opportunity to use such extensive research of environmental bacterial DNA from the mine water yet, we will work on it to improve the determination of bacterial communities in the environment in next coming study.
Point 12: Estimate the concentration of metals is preferring by certain species and estimate the pH affect their vital activity.
Response 12: The microorganisms that was obtain by cultivation, we was testing for their growth on agar with different concentrations of heavy metals, biosorption from the medium also. The results we would published in the next article after ending of all experiments. We have also analyzed the occurrence of heavy metals at the sampling site and the results are part of a forthcoming study.
Author Response File: 
Author Response.docx
Round 2
Reviewer 2 Report
The manuscript has improved a lot. In reference section, some journals are abbreviated while others are not. Check the journal format. English language falters at few places.
Reviewer 3 Report
Thanks for your revision, the quality of this manuscript improved a lot.
