Green Chemistry Based Gold Nanoparticles Synthesis Using the Marine Bacterium Lysinibacillus odysseyi PBCW2 and Their Multitudinous Activities

Round 1

Reviewer 1 Report

1. The abstract should be clearer with detailed findings and results. 

2. Author should more literature background about the green synthesis of nanoparticles. It is suggested to use recent publications. 

a. Ex: https://doi.org/10.1007/s10876-022-02280-z

b. https://doi.org/10.1080/03067319.2022.2079083

c. https://doi.org/10.1016/j.jallcom.2021.162502

d. https://doi.org/10.1016/j.clet.2021.100227

3. There is a lack of literature regarding dye degradations and antibacterial activity in the introduction sections. It must be improved.

 

4. You have typo mistakes of suffixes in the chemical formula in different places. You can check line 75; here are the mistakes of AgNO3. Therefore, it is recommended to carefully check the full manuscript and correct it accordingly. 

5. When using different testing procedures, please use the appropriate references. 

6. The reference styles are different in different places. Such as in Line 285, you have used (Lee et al., 2004). Which is wrong citation styles for this journal. Please check carefully and follow the citation styles of MDPI. 

7. In figure 3 (b), The X-axis has no values.

8. What was the thickness of your synthesized gold nanoparticles? 

9. Comparing your antibacterial result with previously published papers is better. 

10. The author needs to demonstrate the fitted line in the kinetic study during the dye degradation. https://doi.org/10.1016/j.inoche.2020.108156

 

Author Response

Response to Reviewer’s Comments

Title: Green chemistry based gold nanoparticles synthesis using marine bacterium Lysinibacillus odysseyi PBCW2 and their multitudinous activities.

Reviewer 1

1. The abstract should be clearer with detailed findings and results.

• We agree with the concern and now the abstract has been rephrased.

2. Author should more literature background about the green synthesis of nanoparticles.

• We agree with the comment and we have included additional references to the relevant text in the revised manuscript.

3. There is a lack of literature regarding dye degradations and antibacterial activity in the introduction sections. It must be improved.

• We have improved the introduction part by adding the proper literature references dealing with dye degradation and antibacterial activity.

4. You have typo mistakes of suffixes in the chemical formula in different places. You can check line 75; here are the mistakes of AgNO₃. Therefore, it is recommended to carefully check the fullmanuscript and correct it accordingly.

• We are thankful for these important suggestions and the necessary changes have been made throughout the manuscript.

5. When using different testing procedures, please use the appropriate references

• We appreciate the concern. Only the relevant references have been cited.

6. The reference styles are different in different places. Such as in Line 285, you have used (Lee et al., 2004). Which is wrong citation styles for this journal. Please check carefully and follow the citationstyles of MDPI.

• The reviewer concern is well taken. Necessary changes have been made throughout the revised manuscript.

7. In figure 3 (b), The X-axis has no values.

• Reviewer concern is well taken. X-axis indexed.

8. What was the thickness of your synthesized gold nanoparticles? 

• The size of the AuNPs observed from the TEM measurements was found to be between 10 and 100 nm whereas the thickness of the NPs cannot be measured, as it is a single dimensional structure. We made some attempts, but we could not measure the thickness of AuNPs.

9. Comparing your antibacterial result with previously published papers is better.

• Reviewer concern is well taken. Comparative accounts have been mentioned previously.

Reviewer 2 Report

The authors wrote an article on green chemistry-based synthesis of gold nanoparticles using marine bacterium Lysinibacillus odysseyi PBCW2 and investigation of its multitudinous activities. The gold nanoparticles were extracellular synthesized using Lysinibacillus odysseyi PBCW2 bacteria resulting in gold nanoparticle solution dispersed in the cell-free supernatant. The ruby colored solution exhibited an absorbance band around 520 nm confirming presence of gold nanoparticles. Further, the gold nanoparticles were characterized with Electron Microscopy (EM), X-Ray Diffraction (XRD), Dynamic Light Scattering (DLS) and Thermogravimetric Analysis (TGA). The EM showed that the nanoparticles were round-shaped with sizes in the range from 10 to 30 nm. XRD showed presence of an amorphous halo and reflections associated with gold nanoparticles. That result was confirmed with Electron Diffraction. The DLS showed presence of nanoparticles grouped in two modalities with sizes from 1 to 7 nm, and from 10 to 100 nm. The FTIR spectra were recorded for LBOE and LBOE-gold nanoparticles revealing presence of various functional groups. The TGA showed that thermal decomposition of prepared nanoparticles undergoes in three steps described as water loss and organic compound combustion. The resultant samples were found to be perspective for antioxidant, antibacterial and dye degradation applications.

After reading the article, I have an impression that it needs extensive style corrections and advanced data analysis due to the following reasons:

Major:

The introduction section doesn’t provide sufficient information on the topic:

-          No literature review on some classic methods for gold nanoparticle synthesis, e.g., citrate or starch reduction, and its comparison to bacterial synthesis. The advantages of latter approach are not highlighted.

-          No information on advantages for use gold nanoparticle as catalytic or antibacterial species comparing to silver or platinum nanoparticles, nanoclusters or ions. It’s a must especially regarding antibacterial applications as silver dominates in the field.

-          No discussion on the use of various bacteria for gold nanoparticle synthesis. The advantages of Lysinibacillus odysseyi PBCW2 aren’t highlighted.

The data after the characterization wasn’t fully analyzed:

-          In fig. 4 (a), we clearly see an amorphous hallo in the range from 15 to 35 2θ degrees. The halo usually arises due to presence of some amorphous species in the sample, e.g., hydroxides or hydroxy oxides.

-          Fig. 4 (b) shows some small nanoparticles and no histogram with the size distribution. The histogram for at least 500 nanoparticles is necessary.

-          Fig. 4 (c) shows the Electron Diffraction pattern. However, the pattern isn’t indexed.

-          Fig. 5 (a) shows the elemental composition. The spectrum is quite noisy. It’s unclear why the sample contains a lot of zinc, potassium, sodium and chlorine.

-          Fig. 5 (c) shows the results of Thermogravimetric analysis (TGA). The authors don’t comment on possible hydroxide decomposition in the sample. Did the water loss happen due to that reason? Was the sample analyzed with XRD after TGA? I’m curious about the amorphous halo.

-          The DLS analysis revealed two distinct collections of nanoparticles with sizes from 1 to 7 nm, and from 10 to 100 nm. This result contradicts with the data obtained with Electron Microscopy.

Minor:

-          ABSTARCT and CONCLUSIONS sections are too long. The abstract should be no longer than 200 words. The conclusions remind the abstract too much.

-          The synthetic approach isn’t clearly described in the text. In particular, the “2.4 Synthesis, optimization and yield of LBOE-AuNPs” doesn’t fully describe the “minor modifications, process optimization and standardization”. Likewise, it’s unclear from the description what the Lysinibacillus odyssey extract was and how it was produced.

-          The section “2.5 Characterization of gold nanoparticles” doesn’t provide sufficient information on the characterization techniques and its usage. It’s merely a list of equipment.

-          Fig. 1 and 2 show the absorbance of gold nanoparticle solution. The background signal is incredibly strong. It’s unclear why. Likewise, as the multiple peaks are visible in the spectra, it’s necessary to conduct the fittings and show the analyzed data in the table.

-          The mechanism of gold nanoparticle antibacterial activity isn’t clearly described. Are the prepared gold nanoparticles better than silver ions, nanoclusters and nanoparticles?

STYLE, DATA PRESENTATION and CITATIONS:

-          Over the article the multiple citations are present in format “[1][2]” “[3][4][5]”, e.g., page 2 line 53 or page 2 line 58, rather than [1, 2] or [3-5]. That definitely needs to be edited out.

-          Fig. 1 and 2 represent quite similar data. Is it possible to combine those two figures in the single?

-          The article definitely suffers from the absence of a Discussion section.

-          It’s better not use colored frames around the figures.

Therefore, I cannot recommend the article for publication in the present state. The article needs major revision.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear Authors,

I have gone through the revised manuscript. You paid a lot of attention to the Introduction part. That lifts away my first question raised in the major revision. However, the second question regarding sample characterization and sample analysis isn’t fully addressed, as I cannot find clear answers in the revised text. First of all, I’m asking you to answer the question with clear reference to the text indicating page and line. That significantly simplifies my work.

Regarding the second question in the major revision:

-          Your answer on “amorphous” halo doesn’t fully address my question. As far as I know gold complex compounds do crystallize and, subsequently, give X-ray reflections in the pattern. Likewise, no information on spectroscopic evidence of those compounds is present in the article. I mean that amorphous halo cannot be a finger print for the complexes without other supporting data. Please discuss other possible causes for halo observation too.

-          I cannot accept your refuse to show a size histogram for nanoparticles shown in the Figure 4 (b). The reason is that the image shows nanoparticles with diameters ~15-20 nm inconsistent with DLS result shown in Fig. 5 (d). Please provide the histogram or the nanoparticle images showing bimodal size distribution. Likewise, the scale bar is poorly visible in the image.

-          That wasn’t mentioned in my first review but the visibility of Electron Diffraction pattern is quite poor. The dark field occupies the majority of image’s area. Please enlarge the area with the pattern. After that please clearly index the pattern.

-          No comments on noise in the EDX signal. Likewise, it’s unclear why there were some admixtures if you washed out your samples before the analysis.

-          I didn’t see the XRD after TGA. Please provide it.

-          The DLS result still shows bimodal size distribution. Please address the question taking into account your Electron Microscopy.

STYLE:

-          The abstract is still too long. It’s definitely longer than 200 words.

-          Before I recommended to provide citations in the style [1-3] but not [1][2][3]

-          Please provide the respective citations in the “2.5 Characterization of gold nanoparticles”, against each instrument if the characterization has already been described elsewhere.

-          Fig. 1 and 2 can be combined into a single one. Please do emphasize the single peak absorption within single figure.

Author Response

Reviewer comments and responses

1. Your answer on “amorphous” halo doesn’t fully address my question. As far as I know gold complex compounds do crystallize and, subsequently, give X-ray reflections in the pattern. Likewise, noinformation on spectroscopic evidence of those compounds is present in the article. I mean that amorphous halo cannot be a finger print for the complexes without other supporting data. Please discuss other possible causes for halo observation too.

• We appreciate the concern. As mentioned by the reviewer that the “gold complex compounds do crystallize and, subsequently, give X-ray reflections in the pattern”, indeed the XRD pattern of the present study has exhibited distinct and specific absorption peaks confirming the crystalline nature of synthesized AuNPs. With regard to the amorphous halo, in continuation with previous response, the halo region may be due to the reduction in size due to lost long-range crystalline order, without complete transition to a specific form (Deng et al., 2008; Yang et al., 2010). The XRD pattern of nano-crystals may also be material-specific and related to the crystalline microstructure (crystal size, micro-strain and defects) of individual substances (Deng et al., 2008). Another explanation for this halo formation may also be the longer crystallization time, as propounded by Xiao et al. (2012); Christodoulouet al. (2019).

2. I cannot accept your refuse to show a size histogram for nanoparticles shown in the Figure 4 (b). The reason is that the image shows nanoparticles with diameters ~15-20 nm inconsistent with DLS resultshown in Fig. 5 (d). Please provide the histogram or the nanoparticle images showing bimodal size distribution. Likewise, the scale bar is poorly visible in the image.

• We agree and initially we thought that the diameter graph would suffice as response to the initial concern. Now, after seeing your comment, the histogram for the particle size distribution with reference to respective TEM image is added in Supplementary file 1.

3. That wasn’t mentioned in my first review but the visibility of Electron Diffraction pattern is quite poor. The dark field occupies the majority of image’s area. Please enlarge the area with the pattern. Afterthat please clearly index the pattern.

• Reviewer’s concern has been taken well. The EDX has been enlarged and indexed.

4. No comments on noise in the EDX signal. Likewise, it’s unclear why there were some admixtures if you washed out your samples before the analysis.

• We appreciate the concern. The sample contains elements other than gold, which may be due to the culture medium used for referenced bacterial species. The pattern was recorded at the time of AuNPs production and before purification.

5. I didn’t see the XRD after TGA. Please provide it.

• We agree with your comments and we apologize that no XRD was recorded after TGA. We request you please kindly consider it for our future communications.

6. The DLS result still shows bimodal size distribution. Please address the question taking into account your Electron Microscopy.

• We appreciate the concern. Contrasting text has been rectified.

7. The abstract is still too long. It’s definitely longer than 200 words.

• Reviewer concern is well taken. Abstract rephrased. Number of words: 144

8. Before I recommended to provide citations in the style [1-3] but not [1][2][3].

• Reviewer concern is well taken. Now in the revised manuscript the recommended style has been formatted.

9. Please provide the respective citations in the “2.5 Characterization of gold nanoparticles”, against each instrument if the characterization has already been described elsewhere.

• We appreciate the concern. Respective citations have been added.

10. 1 and 2 can be combined into a single one. Please do emphasize the single peak absorption within single figure.

• We appreciate the concern. Figures 1 and 2 have therefore been combined into one figure as Fig. 1.