Production, Purification, and Characterization of Bacillibactin Siderophore of Bacillus subtilis and Its Application for Improvement in Plant Growth and Oil Content in Sesame

Round 1

Reviewer 1 Report

Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria capable of enhancing crop plant growth and health.  Bacillus spp. are one of the common soil bacteria well known for their abilities to promote plant growth through various direct and indirect mechanisms. Bacterial production and utilization of siderophore to concentrate the unavailable form of soil iron is of agroecological significance.

The manuscript titled – Production, Purification, and Characterization of Bacillibactin Siderophore of Bacillus subtilis and its application for improvement in plant growth and oil content in sesame by Nithyapriya et al. describes isolation and characterization of plant growth promoting bacteria (Bacillus subtilis LSBS2) from the rhizosphere of sesame. The manuscript also presents purification and characterization of Bacillibactin siderophore produced by the bacteria.

Overall, the manuscript describes comprehensive research work on sesame rhizosphere bacteria Bacillus subtilis LSBS2 and its Bacillibactin siderophore. The manuscript is of high relevance for exploration, efficient PGPR bacterial strain selection and development for new bioinoculants for crop improvement.

Major concerns:

1. In section 2.10 (Plant Growth-Promotion Studies Under Greenhouse Conditions) the Treatment T0 is described as Control without LSBS2 application and treatment T1 as with 200 mL of B. subtilis LSBS2 broth. The experiment plan lacks a Control with plain nutrient broth without the bacteria.
2. In Table 5 (Measurement of nutrients in rhizospheric and non-rhizospheric soil of sesame plants 60 days after seeding with or without B. subtilis LSBS2), and the results section (L 475 – 483) - How can you account for the differences in concentration of soil nutrient elements tested between the rhizosphere soil and the non-rhizosphere soil? The soil that was used in the greenhouse experiment was a mixture of garden soil, sand, and manure was mixed in the ratio 239 of 2:1:1 and sterilized by autoclaving. Change in the concentration of available form of nutrient element is a possibility but not in the total concentration in the same soil.
3. To improve manuscript quality authors need to carefully proof-read and eliminate issues related to grammar, phrasing, and proper technical terms. Few examples below:

L 26 -27          Siderophores are low molecular weight secondary metabolites produced by microorganisms under low stresses of iron (LOW IRON STRESS?) as a specific iron chelator for the iron nutrition of microorganisms.

L53 – 54          Thus, the sesamum indicum L. plays a crucial role within the final seed yield.

 

Author Response

Reviewer 1 Report

Changes in the MSS are highlighted in red font

Overall, the manuscript describes comprehensive research work on sesame rhizosphere bacteria Bacillus subtilis LSBS2 and its Bacillibactin siderophore. The manuscript is of high relevance for exploration, efficient PGPR bacterial strain selection and development for new bioinoculants for crop improvement.

Major concerns

• In section 2.10 (Plant Growth-Promotion Studies Under Greenhouse Conditions) the Treatment T0 is described as Control without LSBS2 application and treatment T1 as with 200 mL of B. subtilis LSBS2 broth. The experiment plan lacks a Control with plain nutrient broth without the bacteria.

 

Authors response : Agreed. T0 is now revised as control with uninocluated NB

 

• In Table 5 (Measurement of nutrients in rhizospheric and non-rhizospheric soil of sesame plants 60 days after seeding with or without B. subtilis LSBS2), and the results section (L 475 – 483) - How can you account for the differences in concentration of soil nutrient elements tested between the rhizosphere soil and the non-rhizosphere soil? The soil that was used in the greenhouse experiment was a mixture of garden soil, sand, and manure was mixed in the ratio 239 of 2:1:1 and sterilized by autoclaving. Change in the concentration of available form of nutrient element is a possibility but not in the total concentration in the same soil.

 

Authors response : The table 5 is now revised. Data of soil nutrients in untreated soil and LSBS2 treated soil was given.

• To improve manuscript quality authors need to carefully proof-read and eliminate issues related to grammar, phrasing, and proper technical terms. Few examples below:

 

• L 26 -27- Siderophores are low molecular weight secondary metabolites produced by microorganisms under low stresses of iron (LOW IRON STRESS?) as a specific iron chelator for the iron nutrition of microorganisms.

 

• L53 – 54 -Thus, the sesamumindicum L. plays a crucial role within the final seed yield.

 

Authors response : The manuscript has been proofread and improved. The issues related to grammar, phrasing, and proper technical were eliminated

L 26 -27- Agreed and corrected as low iron stress [Line No. 27].

L53 – 54 – This sentence is now deleted

Author Response File: Author Response.pdf

Reviewer 2 Report

The present manuscript is a research work in the field of plant growth-promoting bacteria. The study focuses on producing pure and characterized siderophores from Bacillus strains that are efficient in stimulating plant growth, oil content increase, and general development of sesame plants.

Generally, the manuscript is well-organized according to the Instructions to authors, but in some places that are mentioned in continuation, it has to be improved.

The abstract needs to be rewritten to define the most valuable from the study and be shortened to compile the Sustainability requirements. Mainly, it is not clear how the authors concluded that inoculation of Bacillus subtilis LSBS2 results in improved soil microbial populations (l. 39). It's not clear.

The manuscript includes an Introduction section that has to be improved adding more information about the approach used by the authors with addition of broth suspension of B. subtilis isolate and not cell harvesting by centrifugation, washing, resuspension, etc. Also, more information about the addition of pure siderophore suspension and how this corresponds to the literature and to use the control irrigated only with water.

In general, the manuscript is too heavy and needs to be rewritten for clarity and readability.

The objective of the present study described in lines 81-85 needs rewriting. The authors did not synthesize the questions very well on the basis of which they developed the present study.

In l. 112 the subtitle needs to be revised.

The section Material and Methods is too large and needs to be shortened. Try to use references without unnecessarily describing the methodological procedure. A clear example of this is section 2.3, where the authors described a very routine procedure in detail.

The sentence between l.153 and l.155 needs revision. In this sense, the whole manuscript has to be checked for clarity of sentences, paragraphs, meaning and sections. In continuation of the above mentioned, the first paragraph of section 2.5 (l.136-138) also is unclear and needs revision.

In the design of greenhouse experiment, the difference between T0 and T1 is the application in the last one of broth medium together with B. subtilis population. It was necessary to add bacterial population without broth because you have introduced nutrients to the soil, thus the difference between treatments is not only the Bacillus subtilis population. So, there is a different source of nutrients that influence for sure the results. If no explanation, it seems to be a methodology error.

In l. 311, the reference used is not the most appropriate for this type of analysis. In general, the viable cell count is a simple study of soil microorganisms' behavior, especially their populations, to be used alone.

The titles of all pictures in Figure 1 need to be checked, some of them rewritten or other picture to be changed to be clearer.

I think that the Results of section 3.8 are erroneous and unnecessary. While the treatment T1 is erroneous if there is added broth medium together with the bacterial population.

The authors should revise whole the list of references and use one single way to describe them, with or without DOI number.  

Author Response

Reviewer 2 Report

Corrections/Revision is highlighted in blue fonts.

Generally, the manuscript is well-organized according to the Instructions to authors, but in some places that are mentioned in continuation, it has to be improved.

• The abstract needs to be rewritten to define the most valuable from the study and be shortened to compile the Sustainability requirements. Mainly, it is not clear how the authors concluded that inoculation of Bacillus subtilisLSBS2 results in improved soil microbial populations (l. 39). It's not clear.

Authors response: The abstract is rewritten and shortened to 12 lines from 18 lines. Data regarding soil microbial populations has been removed.

• The manuscript includes an Introduction section that has to be improved adding more information about the approach used by the authors with addition of broth suspension of  subtilisisolate and not cell harvesting by centrifugation, washing, resuspension, etc. Also, more information about the addition of pure siderophore suspension and how this corresponds to the literature and to use the control irrigated only with water.

Authors response: Information on plant growth promotion by Bacillus is already given in introduction. However, some additional information is also provided.More information about the addition of pure siderophore suspension and how this corresponds to the literature is also mentioned. . [Line No 56, 65-68]

• In general, the manuscript is too heavy and needs to be rewritten for clarity and readability.

Authors response: The manuscript has been revised precisely and reduced to 8195 words (18 pgs) from 9591words (20 pgs).

• The objective of the present study described in lines 81-85 needs rewriting. The authors did not synthesize the questions very well on the basis of which they developed the present study.

Authors response: The objectives of the present study are revised. [Line No 70-73]

• In l. 112 the subtitle needs to be revised.

Authors response: The subtitle has been revised.

• The section Material and Methods is too large and needs to be shortened. Try to use references without unnecessarily describing the methodological procedure. A clear example of this is section 2.3, where the authors described a very routine procedure in detail.

Authors response: The materials and methods have been shortened and revised. [Line No 92]

• The sentence between l.153 and l.155 needs revision. In this sense, the whole manuscript has to be checked for clarity of sentences, paragraphs, meaning and sections. In continuation of the above mentioned, the first paragraph of section 2.5 (l.136-138) also is unclear and needs revision.

Authors response: The above-mentioned sentences and sections have been revised and changed.

• In the design of the greenhouse experiment, the difference between T0 and T1 is the application in the last one of broth medium together with B. subtilis population. It was necessary to add bacterial population without broth because you have introduced nutrients to the soil, thus the difference between treatments is not only the Bacillus subtilis population. So, there is a different source of nutrients that influence for sure the results. If no explanation, it seems to be a methodology error.

Authors response: T0 was the uninoculated NB, as NB was used as a medium to grow the bacteria. So its effect was studied

• In l. 311, the reference used is not the most appropriate for this type of analysis. In general, the viable cell count is a simple study of soil microorganisms' behavior, especially their populations, to be used alone.

 

Authors response: Reference No. 14 is revised.

• The titles of all pictures in Figure 1 need to be checked, some of them rewritten or other pictures to be changed to be clearer.

Authors response: The title of all pictures in Figure 1 has changed.

• I think that the Results of section 3.8 are erroneous and unnecessary. While the treatment T1 is erroneous if there is added broth medium together with the bacterial population.

Authors response: The results of section 3.8 are removed.

• The authors should revise whole the list of references and use one single way to describe them, with or without DOI number.  

Authors response: The whole list of references has been described in a single way.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The abstract is still more than 200 words despite rewriting.

Section Introduction was not improved, as I mentioned.

As I mentioned in the first review, the objective of the present study described in lines 81-85 needs rewriting. The authors did not synthesize the questions very well based on which they developed the present study. The authors did not take into account that, so there are factual errors that they must remove.

Most of my comments are not reflected in the revised version.

Author Response

Response to Comments of Reviewer 2 Round 2

The authors are very much thankful to the reviewers for the suggestions that helped in improving the MSS

• The abstract is still more than 200 words despite rewriting.

Authors’ response: The abstract is only 163 and not more than 200 words

• Section Introduction was not improved, as I mentioned.

Authors’ response: Introduction section was improved as per the suggestion. Information on plant growth promotion by Bacillus is mentioned. More information about the addition of pure siderophore suspension and how this corresponds to the literature is also mentioned [Line No 56, 65-68].

• As I mentioned in the first review, the objective of the present study described in lines 81-85 needs rewriting. The authors did not synthesize the questions very well based on which they developed the present study. The authors did not take into account that, so there are factual errors that they must remove.

Authors’ response: The objectives of the present study are now revised [Line No 70-74]

• Most of my comments are not reflected in the revised version.

Authors’ response: All the corrections as suggested by the reviewer in Round 2 have been incorporated and highlighted in blue fonts.

Reviewer’s previous comments and Authors Response

• The abstract needs to be rewritten to define the most valuable from the study and be shortened to compile the Sustainability requirements. Mainly, it is not clear how the authors concluded that inoculation of Bacillus subtilisLSBS2 results in improved soil microbial populations (l. 39). It's not clear.

Authors response: The abstract is rewritten and shortened to 163 words. Data regarding soil microbial populations has been removed.

• The manuscript includes an Introduction section that has to be improved adding more information about the approach used by the authors with addition of broth suspension of  subtilisisolate and not cell harvesting by centrifugation, washing, resuspension, etc. Also, more information about the addition of pure siderophore suspension and how this corresponds to the literature and to use the control irrigated only with water.

Authors response: Information on plant growth promotion by Bacillus is already given in introduction. However, some additional information is also provided. More information about the addition of pure siderophore suspension and how this corresponds to the literature is also mentioned. . [Line No 56, 65-68]

• In general, the manuscript is too heavy and needs to be rewritten for clarity and readability.

Authors response: The manuscript has been revised precisely and reduced to 8195 words (18 pgs) from 9591words (20 pgs).

• The objective of the present study described in lines 81-85 needs rewriting. The authors did not synthesize the questions very well on the basis of which they developed the present study.

Authors response: The objectives of the present study are revised. [Line No 70-74]

• In l. 112 the subtitle needs to be revised.

Authors response: The subtitle has been revised.

• The section Material and Methods is too large and needs to be shortened. Try to use references without unnecessarily describing the methodological procedure. A clear example of this is section 2.3, where the authors described a very routine procedure in detail.

Authors response: The materials and methods has been shortened and revised. [Line No 92]

• The sentence between l.153 and l.155 needs revision. In this sense, the whole manuscript has to be checked for clarity of sentences, paragraphs, meaning and sections. In continuation of the above mentioned, the first paragraph of section 2.5 (l.136-138) also is unclear and needs revision.

Authors response: The above mentioned sentences and sections have been revised and changed.

• In the design of greenhouse experiment, the difference between T0 and T1 is the application in the last one of broth medium together with B. subtilis population. It was necessary to add bacterial population without broth because you have introduced nutrients to the soil, thus the difference between treatments is not only the Bacillus subtilis population. So, there is a different source of nutrients that influence for sure the results. If no explanation, it seems to be a methodology error.

Authors response: T0 was the uninocualted NB, as NB was used as a medium to grow the bacteria. So its effect was studied

• In l. 311, the reference used is not the most appropriate for this type of analysis. In general, the viable cell count is a simple study of soil microorganisms' behavior, especially their populations, to be used alone.

Authors response: Reference No. 14 is replaced.

• The titles of all pictures in Figure 1 need to be checked, some of them rewritten or other pictures to be changed to be clearer.

Authors response: The title of all pictures in Figure 1 has Revised.

• I think that the Results of section 3.8 are erroneous and unnecessary. While the treatment T1 is erroneous if there is added broth medium together with the bacterial population.

Authors response: The results of section 3.8 are removed.

• The authors should revise whole the list of references and use one single way to describe them, with or without DOI number.  

Authors response: The whole list of references has been described in a single way.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript still lacks fundamental evidence and experimental descriptions that are required to support the authors’ claims.

 

 

Comment #1

Page 8. The authors need to carefully describe the procedure and present data as to how they purify the substance that they claim bacillibactin-type siderophore. It seems that the manuscript completely lacks scientific evidence in purification and identification of the chemical. Moreover, the authors are asked to show that the application of the purified bacillibactin-type siderophore promotes the growth of sesame plants.

 

Authors’ response: The procedure for purification of siderophore has been described in Materials and Methods under the heading 2.8. Purification of siderophore on Amberlite XAD-400 resins (Line 181-195) and the data on purification is presented in Results section 3.5.1. Purification on Amberlite XAD-400 resins (Line 341-350).

 

Comment to the authors’ response

The revised manuscript still lacks core information of how exactly the authors performed the purification of the siderophore.

 

342. I am wondering whether ‘XAD-400 resin’ (lines 33, 180, 185, 189, 340, and 486) is indeed purchasable from Sigma or should be read as ‘XAD-4’ as described in line 342. The choice of resins in purifying small molecules should affect the interpretation of virtually all the results of the manuscript.

https://www.dupont.com/content/dam/dupont/amer/us/en/water-solutions/public/documents/en/45-D00733-en.pdf

1. How much amount of starting material was required for them to purify what they claim bacillibactin-type siderophore, and how much amount of the siderophore was fractionated in each of the fraction in Table 2?

• The authors also need to present the purification fold as well as the recovery rate of the siderophore relative to the starting material. These values are essential not only in evaluating the efficiency of the purification of the siderophore, but in reproducing/repeating the whole experiments when necessary.

2. The authors should present UV-Vis spectra of 5 fractions in Table 2.
3. I have no clue of what the authors wish to claim “No extraction was obtained with any of the solvents used.” (line 341).
4. The authors need to clearly describe which of the fractions in Table 2 were subjected to TLC (line 350) and show photos of TLC plates.

• I doubt “a red color spot” (line 352) that the authors observed after spraying was not the only substance that existed in the fraction. This is especially when the resin that the authors used was XAD-4, which technically absorbs anything in the extract. Therefore, the authors should indicate how exactly they further purified, if any, the fractions that they subjected to TLC prior to FT-IR analysis.
• The revised manuscript lacks the response to the following original comment “Moreover, the authors are asked to show that the application of the purified bacillibactin-type siderophore promotes the growth of sesame plants.”. While the authors show in Fig. 8 and Table 3 that the application of subtilis LSBS2 affects the growth of S. indicum, I am still not convinced that bacillibactin in this particular strain of B. subtilis is responsible for the growth alteration of S. indicum. The authors need to show that a) the application of a mutant strain of B. subtilis LSBS2 deficient in bacillibactin production does not affect the growth of S. indicum and that b) application of pure bacillibactin affects the growth of S. indicum plants.

 

 

Comment #2

Since the authors only investigated Bacillus spp. in the metagenomic analysis, there are no ways of testing whether microorganisms other than Bacillus spp. might also contribute to iron acquisition of sesame plants as presented in Table 1. This in turn relates to the feasibility of the proposed link between the increased level of iron in rhizosphere soil from Sesamum compared to non-rhizosphere soil (Table 1) and the growth promotion of sesame plants when inoculated with B. subtilis LSBS2 (Tables 2 to 4). Bacillus spp. and the siderophore.

 

Authors’ response: The garden soil used for pot assay was sterilized to eliminate the microorganisms present in the soil (Line 225).

 

Comment to the authors’ response

I confirmed that the use of sterilized soil was described in the original manuscript.

 

 

Comment #3

Fig. 3. Apparently, either or both the choice of column or the solvents should be carefully optimized so that the peaks are well retained and resolved. Representative chromatograms of crude extracts and purified fractions should be presented.

 

Authors’ response: The column and solvent were chosen based on the standard methodologies [Reference No 34, 35]. Only purified fraction peaks are retained in the manuscript.

 

Comment to the authors’ response

The authors claimed in line 380 that “The peaks appeared at 1-0.767 min, 2-2.033 min, using was used as a standard”. However, there are no data showing these peaks of standards. Moreover, the peaks in the chromatogram at 220 nm (Fig. 3) were not well separated, and all the peaks at Rt 1.621, Rt 2.188, and Rt 2.473 have shoulders thus apparently include more than two compounds, respectively. The authors are asked either to co-chromatograph the fraction with bacillibactin standard or to at least provide a chromatogram of the standard and compare their Rt. The authors should clearly indicate which of the peak was bacillibactin as they claimed.

 

 

Comment #4

Line 401. Charts for 2D-NMR should be presented.

 

Authors’ response : Agreed to the comment of Reviewer but we performed only NMR and not 2D-NMR. Requisite corrections have been done [Line 212, 389].

 

Comment to the authors’ response

I am wondering whether and how the authors confirmed the purity of the extract prior to NMR analysis. The authors should also present NMR data of standard bacillibactin.

Th

 

Comment #5

Fig. 5. The authors should clearly state in which developmental stage the photos were taken. Scale bars required.

 

Authors’ response : Photos were taken at 60 days after inoculation, scale bar photos was added in the manuscript [Line 415-416].

 

Comment to the authors’ response

There still no scale bars in the manuscript.

Reviewer 2 Report

The manuscript “Production, Purification, and Characterization of Bacillibactin Siderophore of Bacillus subtilisand its application for improvement in plant growth and oil content in sesame”, it is interesting to improvement plant growth by using siderophore producing B. subtilisstrain. However, I think that the identification of bacillibactin of this strain is not acceptable because the data seems to be incomplete as shown below.

 

1) In HPLC data, the peaks overlap and the peaks are not strictly separated. The method should be improved.

 

2) The MS spectral data is important as the first step in substance identification of bacillibactin. MS spectral data is necessary to determine the molecular formula and molecular weight of a substance.

 

3) Zhou et al. (2018, Mar. Drugs, 16, 22; doi:10.3390/md16010022) reports that there are several types of bacillibactin. They have published NMR spectrum, IR spectrum, and MS spectrum for those substances, but their NMR spectral data and yours NMR spectral data is not similar.

 

4) In particular, the integrated values of the NMR analysis is questionable, and do not agree with the molecular structure formula shown in Figure 7. Your data is not clear.

For example, the ¹H NMR spectrum of bacillibactin has a feature that includes several H₃split peaks in Zhou et al., (2018), but it is not observed in your data.

 

5) Not enough description and data in the whole manuscript are presented to determine the structure of bacillibactin.

 

 

I would recommend to carefully revise the manuscript, before it can be elsewhere published, according to the following suggestions:

 

All pictures (Fig. 1.1–1.5 and Fig. 8) in the manuscript are not clear. It needs to be changed to clearer (higher resolution) picture.

 

Line 176-176: 50-AGAGTT......AG-30 and other => 5'-AGAGTT......AG-3'

Line 388-389: In 1H NMR aliphatic....(Figure 5) => (Figure 6) ?

Line 394-396: In 13C NMR...... (Figure 6) => (Figure 5) ?

Table 5: Significant difference *, **, ***= extent of significance LSD (P<0.05) => (P<0.05, 0.01, 0.001) ?