Bacteria Isolated from Wastewater Irrigated Agricultural Soils Adapt to Heavy Metal Toxicity While Maintaining Their Plant Growth Promoting Traits

Round 1

Reviewer 1 Report

This review presents a complete study that deals with the widespread problem of agricultural soils polluted with heavy metals. Authors have isolated many different bacteria strains from these contaminated soils, irrigated with wastewater from different origins. They have analyzed their ability to solubilize Zn, K and PO₄ and to produce siderophores and proteases. Moreover, they have studied their ability of fixing nitrogen using Nbf medium and they have detected by PCR nif genes that are critical in this process. In addition of these plant growth promoting (PGP) features, they have analyzed their resistance to different heavy metals (Ni, Pb and Cd) and they have calculated MIC values. Best candidates for bioremediation and biofertilization were identified by 16S rRNA gene sequence analysis and their heavy metal removal ability was defined.

The manuscript is properly written and all the results are clearly explained, being very easy to read and understand it. From my point of view, this study offers a really interesting study and promising results for future studies related to bioremediation. However, I believe that some considerations/corrections should be taken into account by authors before accepting this paper (minor revision):

• Line 25: “Enterobacter also removed 87%, 79% and 43% and 86%, 78% and 51% and of Ni, Cd and”: underlined word is not needed
• References are not correctly ordered along the text. They must be numbered according to their appearance along the text and they are totally aleatory in the present manuscript.
• Line 55: “producing iron chelator (biotransformation)”. In my opinion, biotransformation is a more general concept. Maybe the text could be changed: transforming them in less or non-toxic forms (biotransformation)
• Lines 141-142, line 245 and line 253: “Minimum inhibitory concentration (MIC) of above-mentioned heavy metals for bacterial isolates having PGP ability”, “Bacterial isolates having PGP potential also showed resistance to various concentration of selected heavy metals” and “Bacterial strain having good PGP and heavy metal resistant score were selected for their heavy metal removal ability”.

How were selected bacteria with PGP activity? Criteria for selecting them must be detailed in the manuscript

• Line 160-161: “Control sets, without added bacterial cells were used to compare the metal uptake by bacteria.” Which values were obtained for controls sets in heavy metal removal experiments? Some metal cations may be chelated by the organic matter of the media (LB broth). These values do not appear in the results section and I consider that they are important to really quantify the amount removed by bacteria in the different assays performed.
• Line 184: “Amplification was carried out in 25 uL reaction mixture containing 2 uL of template DNA,”. It might be important to indicate the DNA concentration used in PCR reactions
• Table 2: this table also appears in supplementary material (S3). It should be maintained only in the manuscript and be eliminated from the supplementary material. In this way, the rest of the supplementary tables must be renumbered.

In PCR conditions column, only Tm will be necessary to appear as the rest of the information is the same for all the primers pairs. A better option could be to write this information in the text and only write Tm in the last column of this table.

• Line 248: Bacterial strain FWM9”: there is a mistake in this line, it is referred to FWN9 strain
• Line 253: How was calculated the heavy metal resistant score? This information should be added to material and method section
• Line 264-265: “Bacterial isolates belonged to various genera i.e., Bacillus, Pseudomonas, Enterobacter, 264 Citrobacter, Acinetobacter, Serratia, Klebsiella and Enterococcus (Table 3).” The percentages of each genera could be added to the text in order to highlight the most abundant ones. Strains from the genera Escherichia also appears in the analysis and they are not mentioned in the text. You should include it in the list.
• Table 3: this table does not include all the isolated strains, only the ones with PGP potential and heavy metal tolerance. You should mention it at the bottom of the table (similarly to Fig 4).
• Fig 4: a higher number of strains is included in this analysis than the ones that are compiled in table 3 but they may be the same, right? What is the meaning of red asterisks? You should indicate this information at the bottom of the figure.
• Line 317: “These inorganic acids chelate PO4”: mistake, the correct word is organic
• Table S2: Was not included nitrogen fixation in the scoring criteria?
• Table S5: you should indicate (maybe at the bottom of the table) that these values correspond to the score index defined in Table S2

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present study has investigated the bacteria isolated from wastewater irrigated agricultural soils adapt to heavy metal toxicity while maintaining their plant growth promoting traits. In general, I think the paper is well organized. The subject addressed in this article is worthy of investigation, and it may appeal readership of "Sustainability" journal. In my opinion, this paper is suitable for publication in the journal of sustainability after doing some minor revise. 
1-The manuscript needs a moderate revision for the English Language.
2-There is a lack of connection between paragraphs in the abstract section.
3-The novelty of the paper should be stated clearly.
4-As a suggestion, it would be better to prepare a map instead of Table (1)
5-The descriptions in the conclusion section is not sufficient and should be extended. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The reviewed manuscript sustainability-1243881 presents the results of a painstaking study on the potential of many bacterial strains isolated from wastewater irrigated agricultural soils in Pakistan to promote plant growth and remove heavy metals. The idea of this study is interesting and has international significance because many underdeveloped countries face similar problems. The manuscript is properly organized and written; the methodology is appropriately selected for the planned experiments, the results are valuable although sometimes imprecisely presented in the graphs,  the discussion is short and concise. I read this manuscript with great interest.

The weaknesses of the article concern the following points: 

1. It is customary not to put full stops at the end of sentences in titles.
2. Table 1. Why are there capital letters in words “municipal” and “textile”?
3. Line 112. Clearly express micro (μL).
4. Line 152. please replace form with from.
5. Line 154. 5 × 10⁸ cells mL¹ – or mL^-1? Standardize the notation of units in the text: mg L^-1 or mg/L.
6. Line 181. specie specific primers? Please correct.
7. Table 2 would look nicer if the forward and reverse primers were on single lines.
8. What exactly means the bacterial strain codes? They are quite complicated and can't they be replaced with e.g. numbers?
9. There are many errors in the axis description in Fig. 1 a, b and c. Solubulization Index should be replaced by Solubilization index (OY axis) and Bacterial Strians should be replaced by Bacterial strains. The question is also why are the bacteria codes presented in any order? What is the cause of it? For example LWN4, LWN1, LWN8 (Figure 1a) etc, not LWN1, LWN4, LWN8 order? Moreover, statistical differences between solubilization indices are difficult to interpret, e.g., defgh. Please think about a clearer presentation of all the results in this figure.
10. Similarly, Figure 2 is challenging to analyze. For an elegant presentation, both figures should also be more delicate and similar in size and descriptions.
11. Figure 5 is very large. Maybe you can reduce it and improve the spelling of siderophore production (is sidrophore production).
12. Figure 6 is huge and not very good quality. In addition, the direction of the arrows should be rather the opposite, because the tip shows nothing, only gray.
13. Line 354. Please explain IAA (indole acetic acid) in the discussion section.
14. I suggest you read the text carefully and use the help of a native speaker because many sentences are confusing and have a low scientific level. This will certainly increase its quality and make the message more attractive to the potential reader.

In conclusion, the manuscript requires minor revision and, after correction, should be considered for publishing in Sustainability.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

In my opinion, the article entitled "Bacteria isolated from wastewater irrigated agricultural soils adapt to heavy metal toxicity while maintaining their plant growth promoting traits" is a very interesting study. The article has been written in a very detailed and logical manner. My only attention concerns the lack of information on the quality of wastewater used for soil irrigation - I believe that in the methodology in section 2.1 should take into account the quality of wastewater used for soil irrigation (concentration of organic compounds, nutrients and heavy metals). 

As final general comment, please make sure to define ALL the acronyms form their first appearance in the paper. Also, all the references MUST BE CHECKED and formatted as required by MDPI-Sustainability, also make sure that all the references have DOI number unless it is not available.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf