Peruvian Native Bacterial Strains as Potential Bioremediation Agents in Hg-Polluted Soils by Artisanal Mining Activities in Southern Peru

Round 1

Reviewer 1 Report

General comments

 This is an interesting idea, and the paper has the potential to make a useful contribution to research in this field. Please pay attention to grammar.

Specific comments

 1.       Kindly explain the abbreviation used in the abstract first time.

2. Why it is necessary to conduct this research? what is the hypothesis here? Please rewrite with more clarity

3. Fig. 2: Error bars of standard deviation are missing. Please add.

4. In discussion section, use the findings in this article to discuss, but not summarize the literature. There is need to enhance discussion section.

5. There are little common knowledge in the introduction and discussion. The author should check the whole paper.

6. At some places, sentences are too long. Try to break up these long sentences.

7. Conclusions are just repetition of results. This is not the way to conclude. Please provide some futuristic approaches and rewrite.

 

Author Response

We apologize for the delay in reply to the Review Report. We have rewritten several sections of the manuscript and attempted to corroborate the results presented. We have attached the review file; in this particular case, the Abstract section appears to have not been modified. However, the modifications suggested by the reviewer were made.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The work presented by the authors, entitled "Peruvian Native Bacterial Strains as Potential Bioremediation Agents in Hg-polluted Soils by Artisanal Mining Activities in Southern Peru" addresses a topic of extraordinary importance. Specifically, the need to find environmentally responsible (and effective) alternatives to eliminate or mitigate the presence of metals in soils.

To this end, the authors have isolated two bacterial strains (Kocuria and Zhihengliuella genera) and have evaluated their ability to mobilize Hg in vitro.

Although the hypothesis is valid (although ambitious), the development of the work presents several Achilles heels. Here are some major issues:

1. Point 2.6. from work. To evaluate the capacity of the strains to eliminate Hg, the authors use water + mercurial salts and incubation is carried out up to a maximum of 1080h. I wonder how it is possible that without any nutritional source, bacteria are able to survive such long incubations, even more so in the presence of a stressor, such as Hg. In addition, important data such as the incubation T are missing. Over time, the water evaporates and the Hg concentrates, changing the initial concentration. Authors should detail how they have avoided this situation.

2. L 220-223. The authors emphasize that according to the phylogenetic tree, strain T1.1 could be a new strain because it is on a different branch. Unfortunately, this statement is not subject to any scientific evidence. For environmental strains, as is the case, the sequencing of the 16sRNA gene (or other Housekeeping genes) does not provide enough information to reach such a conclusion. Only the complete sequencing of the genome will be able to shed light on this hypothesis. Nor does the tree itself allow the drawing of such a conclusion. The information in the content should be extended to nearby taxa to see with perspective to which clade it belongs. For all these reasons, the inclusion of phylogeny, in its current form, in all the work is not considered valid.

3. L 243-253. Regarding the antibiogram of the strains, the authors justify that they do so to ensure the biosafety of the strains in subsequent uses in bioremediation processes. This is essential and necessary. However, the authors use the Kirby-Bauer method, which only provides phenotypic information on the S/R profile if there is a valid reference to compare the size of the diameter (zone of inhibition). These references (CLSI, EUCAST,...) are used in the clinical world to characterize strains phenotypically, but they do not include taxa with low clinical incidence (Bacillus spp., for example, still does not have breakpoints and is a better genus characterized by its clinical potential). I wonder what database they have pulled the S/R profiles against. In the absence of verification, I seriously doubt that there are references. This invalidates the entire trial.

4. Table 3. Antibiogram results. There is serious confusion in saying that the MIC value is the first column. What it really is is the load (potency) of the antibiotic. According to the information provided, and in line with the previous comment, the authors have no means of knowing the MIC or the phenotypic resistance profile of the strains. The authors refer to the size of the halo, when it is known that a larger halo does not necessarily correspond to a more sensitive profile (L248-251). Once again, this invalidates the trial. An alternative that the authors have is to sequence the complete genome and, with this, answer the question of whether it is a new species and confirm the existence of functional antibiotic resistance genes or not.

5. The authors do not evaluate the pathogenic potential of the strains. It is an essential requirement to be able to conclude that they are good candidates to mobilize Hg in the environment. Once again, WGS techniques would help to decipher these needs.

 

Minor issues:

1. Lack of uniformity in concepts such as "Gram-positive" sometimes written in capital letters and others in lower case. The authors, eventually, do not adequately cite the bacterial strains and do not adequately refer to their taxonomic level.

2. The units are not properly expressed.

3. The authors incubate at 37ºC. It is rare for environmental strains to have such high optimum growth temperatures.

4. The authors use blood agar for isolation, what is the rationale? Regardless of whether there are hemolytic environmental strains (Bacillus spp., Clostrodium spp.... and many more), its use for environmental strains does not seem logical.

5. In the materials and methods there are clearly discursive parts that must be relocated (L 85-93; L101-103)

6. In the introduction, too much time is spent talking about arid soils, which is not relevant in the development of the research. This part should be reduced in favor of a better explanation of the risks of Hg for human and environmental health.

For all these reasons, the authors are advised to review the content of their manuscript and redesign some experiments, in order to respond to their hypothesis. Also, authors are advised to focus on one of the strains at a time, as the amount of information they want (and can) provide is a lot. By including the two strains, the level of profusion with which it is described has been reduced.

Author Response

We apologize for the delay in reply to the Review Report. We have rewritten several sections of the manuscript and attempted to corroborate the results presented. We have attached the review file.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments to authors:

I have revised your manuscript entitled “Peruvian Native Bacterial Strains as Potential Bioremediation Agents in Hg-polluted Soils by Artisanal Mining Activities in Southern Peru” This is an interesting topic. But it needs some important changes. In select cases, poor grammar and/or word choice may lead to confusion and/or misconceptions; I have marked the most problematic of these in the attached file. Some contradicting information in the data needs to be resolved, and the conclusions were also somewhat simplified; I have also commented on this in the attached file.

·         Units must be according to the International System of Units (SI) in the manuscript.

·         Abbreviations and acronyms should be used consistently throughout the manuscript. Language and grammar should be carefully checked for readability.

·         Linguistic correction in the manuscript is taken into account.

·         The discussion is weak in this manuscript

·         The results must be well discussed

The conclusions section should not be a summary of your study or an extension of the discussion. The conclusions section should illustrate the mechanistic links of findings obtained under applied treatments. The authors should avoid repeating what has already been presented in results and discussion. Please, avoid using abbreviations and acronyms in this section. Remember that the conclusions must be self-explanatory. The conclusions should highlight the novelty and implication of your study.

Comments for author File: Comments.pdf

Author Response

We apologize for the delay in reply to the Review Report. We have rewritten several sections of the manuscript and attempted to corroborate the results presented. We have attached the review file.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have made substantial changes in the manuscript. They have incorporated all the suggestions and now the manuscript looks in good shape.

Reviewer 2 Report

Dear Authors,

I have reviewed the MS again and, in general terms, it has improved compared to the original version. The antibiotic sensitivity part that, as I commented in the original review, was the most "tricky" and has been improved. The part of biosafety in the management of strains is something that the authors must take into account in the future. I understand that WGS is a technique that is still inaccessible to many groups, but for use in the field of this type of microorganism, I consider it necessary. Perhaps not now, but before field trials.

For this reason, I consider the contributions of the authors and the improvements introduced to be valid. I am perfectly aware of the difficulty involved in working with mercury and the limitations that many laboratories have for the safe handling of this element. I believe that the research group is making significant progress and in this new review they have made it clearer that these are first trials and that the objective is to continue advancing in research.

Kind regards,