Microbial-Based Biotechnology: Production and Evaluation of Selenium-Tellurium Nanoalloys

Round 1

Reviewer 1 Report

1. Keywords should be ordered alphabetically

2. Please provide background on the importance of selenium-tellurium nanoalloys and their relevance to drug resistance in the first sentence of the abstract.

3. Please specify the meaning of "B" in Figure 1.

4. Please include the chemical equations for the microbial synthesis of nanoselenium, nanotellurium, and their alloys using sodium selenite and sodium tellurite in the discussion section. Please include the relevant and important bacterial enzyme in the equation.

5. Please provide a detailed description of the metabolism of bacteria involved in the synthesis of nanoselenium, nanotellurium, and their alloys in the discussion section.

6. Bacterial activity, anticancer activity, antibiofilm activity or other relevant activities should be assessed for the fabricated materials synthesized in your research.

Author Response

Dear Reviewer, many thanks for your time and your valuable comments, which really improved our MS, thanks again!

1. Keywords should be ordered alphabetically

Response: done, thanks!

2. Please provide background on the importance of selenium-tellurium nanoalloys and their relevance to drug resistance in the first sentence of the abstract.

Response: Many thanks for your comment!

“Using seleno- and telluric compounds is a practical approach for developing solutions against drug-resistant bacterial infections and malignancies. It will accelerate the search for novel treatments or adjuvants for existing therapies.”

3. Please specify the meaning of "B" in Figure 1.

Response: Many thanks, B refers to the control of Se (without Se) as the tube contains only MRS medium!

4. Please include the chemical equations for the microbial synthesis of nanoselenium, nanotellurium, and their alloys using sodium selenite and sodium tellurite in the discussion section. Please include the relevant and important bacterial enzyme in the equation.
5. Please provide a detailed description of the metabolism of bacteria involved in the synthesis of nanoselenium, nanotellurium, and their alloys in the discussion section.

Response: Many thanks!

Added to the revised MS: the selenite reduction to Se (⁰) in bacterium is intracellular. It is much slower outside. Reducing sugar can convert the selenite to elemental selenium, but that is slow and really nano size. Put selenite to sugar, it will be red after some weeks.  The further reduction could happen depending the redox potential of the solution.

Figure 6: Proposed Mechanism of selenite/tellurite reduction to SeNPs/TeNPs/SeTeNAs

Nanoparticle formation is usually considered as the defence mechanism against metal toxicity. It includes the enhanced excretion or reduced absorption of the metals as their primary response to exposure to high metal concentrations. It includes either reductive precipitation or volatilization of selenite/tellurite [42,43]. A low degree of innate suscepti-bility to SeO32-/TeO32- was exhibited by certain Gram-positive and Gram-negative bacteria [44,45]. Tellurite resistance has been established to be caused by chromosomal (TeR) and plasmid-mediated tellurite resistance (TeR) determinants [44].

Several Enzymatic reductions by bacteria have been reported. Such as thiol or gluta-thione reductase [46,47], sulphite reductase [48], fumarate reductase [49,50], Painter-type reaction [51], and nitrite reductase [52–54]. Depending on the bacterial type, the enzymatic reduction reaction can happen in cytoplasm or periplasm [54]. Taylor et al. [55] explain that the TeO32- enters the cell via the phosphate transporter. Nitrate reductases reduce it [52], where TeO32- can be reduced to Te0. It can also be enzymatically converted to volatile hydride or methyl compounds by cellular reduced thiols. Once selenite/tellurite enters the periplasm, it cannot be used as a distinctive electron acceptor. Reductase enzymes can reduce into Se0/Te0/SeTe-NAs [56].

Figure 6 explains the extracellular and intracellular formation of selenium. In cyto-plasm, Se0/Te0/SeTe-NAs formations are followed by the assembly of Se nanospheres/Te nanorods/SeTe-NAs poly amorphous structures. Intracellular transport mechanisms were exhibited by cells to prevent accumulations of Se nanospheres/Te nanorods/SeTe-NAs [57,58]. T.selenatis transports the Se nanospheres out of the cell from the cytoplasm. It in-cludes the reduction of selenite to Se0 followed by the bounding of SefA protein for assem-bling the Se nanosphere. It is lysed or transported outside the cell by an unknown mecha-nism [59,60]. Many unspecific proteins only interact with intracellular Se-NPs, result-ing in excellent colloidal stability [61].

There is also an extracellular synthesis in the cytoplasm where selenite/tellurite is reduced to Se0/Te0/SeTe-NAs. It is followed by transportation across the membrane as a foreign entity outside the cell by an unknown export mechanism. Then, Se0/Te0/SeTe-NAs nuclei are assembled outside the cell into Se nanospheres/Te nanorods/SeTe-NAs poly amorphous structures through an Ostwald-type ripening mechanism [62,63].

Figure 7: Suggested bacterial reduction of selenite/tellurite reduction to Se⁰/Te⁰/SeTeNAs at the cell surface.

A similar possible mechanism has been reported by Liesje et al. in silver nanoparticle synthesis [64]. Where a) Bacterial cell membrane with reducing sugars such as glucose and protonated anionic functional groups (–RH). b) When pH increases, protons dissociate and create positively charged adsorption sites for SeO₃^2-/ TeO₃^2-. The open-ring structure of reducing sugars can reduce SeO₃^2-/ TeO₃^2-. c) carboxylic acid is formed due to the oxidation of aldehyde group of reducing sugar, while SeO₃^2-/ TeO₃^2- is reduced to Se⁰/ Te⁰/SeTeNAs.

6. Bacterial activity, anticancer activity, antibiofilm activity or other relevant activities should be assessed for the fabricated materials synthesized in your research.

Response: done, thanks!

I have carefully considered your suggestion regarding the assessment of bacterial activity, anticancer activity, antibiofilm activity, and other relevant activities for the fabricated materials synthesized in this study. While I greatly appreciate your input, I would like to inform you that the scope of the current manuscript was primarily focused on those bacteria that couldn’t be able to differentiate selenite or tellurite if we give in certain concentration in the growth media.

However, I am pleased to share with you that these aspects are indeed part of my upcoming research objectives. I am currently in the process of designing experiments that specifically investigate bacterial activity, anticancer properties, and antibiofilm potential of synthesized materials. I believe that this forthcoming work will provide valuable insights into these critical aspects, and I am committed to sharing the outcomes in a subsequent publication.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Author,

The study is impressive. However, I feel that it need more additional experimental design for the hypothesis. I would recommend follow the directions by the editor.

The paper, "Microbial based biotechnology: Production and evaluation of Selenium-Tellurium Nanoalloys" authored by Arjun Muthu and colleagues, offers a good examination. At a foundational level, both Selenium (Se) and Tellurium (Te) are members of the chalcogen group within the periodic table. While they possess overlapping chemical characteristics, their physical and electronic attributes distinctly differ. Merging these elements at the nanoscale gives rise to structures boasting unique electronic, optical, and mechanical features, which are not present in their larger-scale versions. There are numerous techniques available for the synthesis of Se-Te nanoalloys, and several research studies already delve into these Se-Te compounds. In this particular paper, the authors innovatively use the bacterium Lactobacillus casei for the production process. However, the paper seems to narrowly concentrate on the morphological aspects, sidelining other crucial physicochemical properties. It would be beneficial if the authors expanded their research scope to encompass a broader set of parameters and experimental designs.

Author Response

Reviewer 2#

Comments and Suggestions for Authors

Dear Reviewer, many thanks for your time and your valuable comments, which really improved our MS, thanks again!

Dear Author,

The study is impressive. However, I feel that it needs more additional experimental design for the hypothesis. I would recommend follow the directions by the editor.

The paper, "Microbial based biotechnology: Production and evaluation of Selenium-Tellurium Nanoalloys" authored by Arjun Muthu and colleagues, offers a good examination. At a foundational level, both Selenium (Se) and Tellurium (Te) are members of the chalcogen group within the periodic table. While they possess overlapping chemical characteristics, their physical and electronic attributes distinctly differ. Merging these elements at the nanoscale gives rise to structures boasting unique electronic, optical, and mechanical features, which are not present in their larger-scale versions. There are numerous techniques available for the synthesis of Se-Te nanoalloys, and several research studies already delve into these Se-Te compounds. In this particular paper, the authors innovatively use the bacterium Lactobacillus casei for the production process. However, the paper seems to narrowly concentrate on the morphological aspects, sidelining other crucial physicochemical properties. It would be beneficial if the authors expanded their research scope to encompass a broader set of parameters and experimental designs.

Response: done, thanks!

We have taken your suggestion regarding the evaluation of physicochemical properties, including anticancer activity, antimicrobial effects, and other pertinent characteristics of the fabricated materials in this study into careful consideration. While we deeply value your input, we would like to apprise you that the current manuscript's primary focus lies on bacteria that exhibit an inability to differentiate selenite or tellurite when provided in specific concentrations within the growth media.

Nonetheless, we are pleased to inform you that these facets are integral components of my forthcoming research endeavors. We are presently in the process of formulating experiments dedicated to investigating bacterial behavior, as well as exploring the anticancer and antibiofilm potential of the synthesized materials. We are confident that this forthcoming work will yield invaluable insights into these pivotal areas, and we are dedicated to disseminating the outcomes through a subsequent publication.

Added to the revised MS for more improvements to our MS:

The selenite reduction to Se (⁰) in bacterium is intracellular. It is much slower outside. Reducing sugar can convert the selenite to elemental selenium, but that is slow and really nano size. Put selenite to sugar, it will be red after some weeks.  The further reduction could happen depending the redox potential of the solution.

Figure 6: Proposed Mechanism of selenite/tellurite reduction to SeNPs/TeNPs/SeTeNAs

Nanoparticle formation is usually considered as the defence mechanism against metal toxicity. It includes the enhanced excretion or reduced absorption of the metals as their primary response to exposure to high metal concentrations. It includes either reductive precipitation or volatilization of selenite/tellurite [42,43]. A low degree of innate suscepti-bility to SeO32-/TeO32- was exhibited by certain Gram-positive and Gram-negative bacteria [44,45]. Tellurite resistance has been established to be caused by chromosomal (TeR) and plasmid-mediated tellurite resistance (TeR) determinants [44].

Several Enzymatic reductions by bacteria have been reported. Such as thiol or gluta-thione reductase [46,47], sulphite reductase [48], fumarate reductase [49,50], Painter-type reaction [51], and nitrite reductase [52–54]. Depending on the bacterial type, the enzymatic reduction reaction can happen in cytoplasm or periplasm [54]. Taylor et al. [55] explain that the TeO32- enters the cell via the phosphate transporter. Nitrate reductases reduce it [52], where TeO32- can be reduced to Te0. It can also be enzymatically converted to volatile hydride or methyl compounds by cellular reduced thiols. Once selenite/tellurite enters the periplasm, it cannot be used as a distinctive electron acceptor. Reductase enzymes can reduce into Se0/Te0/SeTe-NAs [56].

Figure 6 explains the extracellular and intracellular formation of selenium. In cyto-plasm, Se0/Te0/SeTe-NAs formations are followed by the assembly of Se nanospheres/Te nanorods/SeTe-NAs poly amorphous structures. Intracellular transport mechanisms were exhibited by cells to prevent accumulations of Se nanospheres/Te nanorods/SeTe-NAs [57,58]. T.selenatis transports the Se nanospheres out of the cell from the cytoplasm. It in-cludes the reduction of selenite to Se0 followed by the bounding of SefA protein for assem-bling the Se nanosphere. It is lysed or transported outside the cell by an unknown mecha-nism [59,60]. Many unspecific proteins only interact with intracellular Se-NPs, result-ing in excellent colloidal stability [61].

There is also an extracellular synthesis in the cytoplasm where selenite/tellurite is reduced to Se0/Te0/SeTe-NAs. It is followed by transportation across the membrane as a foreign entity outside the cell by an unknown export mechanism. Then, Se0/Te0/SeTe-NAs nuclei are assembled outside the cell into Se nanospheres/Te nanorods/SeTe-NAs poly amorphous structures through an Ostwald-type ripening mechanism [62,63].

Figure 7: Suggested bacterial reduction of selenite/tellurite reduction to Se⁰/Te⁰/SeTeNAs at the cell surface.

A similar possible mechanism has been reported by Liesje et al. in silver nanoparticle synthesis [64]. Where a) Bacterial cell membrane with reducing sugars such as glucose and protonated anionic functional groups (–RH). b) When pH increases, protons dissociate and create positively charged adsorption sites for SeO₃^2-/ TeO₃^2-. The open-ring structure of reducing sugars can reduce SeO₃^2-/ TeO₃^2-. c) carboxylic acid is formed due to the oxidation of aldehyde group of reducing sugar, while SeO₃^2-/ TeO₃^2- is reduced to Se⁰/ Te⁰/SeTeNAs.

Author Response File: Author Response.docx

Reviewer 3 Report

In this manuscript the authors show how Lactobacilli use selenite and tellurite  to produce nanoparticles. This is an interesting observation with the potential to become clinically important. 

I have a few concerns that should be addressed/discussed before the paper is ready for acceptance.

1. Figure 1 appears to lack a control condition? Could the authors confirm these results by including a heat-killed strain in the reaction?

2. Are these results specific to lactobacilli? I think it would be nice to repeat figure 1 with another pathogenic bacterium to give the paper added clinical weight.

3. Although it is stated in the future work, it would be nice to see a figure with some growth experiments or viability to see if these secreted nanoparticles are antimicrobial towards another bacterium.

4. Did the authors confirm if the secreted nanoparticles are toxic to lactobacilli? 

5. How do the lactobacilli respond to the nanoparticles they secrete? 

Minor issues

1. Sometimes the figures felt a little hard to follow. I suggest making the text and the figures on the same page if possible.

2. The figure legends seem very brief and need a few more details to improve readability. 

3. The EM images in figure 2 appear a bit blurred especially the middle one and if a better quality image could be provided this would be great.

Overall, a nice study with huge potential for the future. 

Author Response

Dear Reviewer,

many thanks for your time and your valuable comments, which really improved our MS, thanks again!

In this manuscript the authors show how Lactobacilli use selenite and tellurite to produce nanoparticles. This is an interesting observation with the potential to become clinically important.

Response: Many thanks for your encouragement! Yes, this unique nano-alloy has distinguished benefits, which can apply in the medicine sector 

I have a few concerns that should be addressed/discussed before the paper is ready for acceptance.

Response: Many thanks, again!

1. Figure 1 appears to lack a control condition? Could the authors confirm these results by including a heat-killed strain in the reaction?

Response: Many thanks, we corrected by adding more information in the figure title

2. Are these results specific to lactobacilli? I think it would be nice to repeat figure 1 with another pathogenic bacterium to give the paper added clinical weight.

Response: Many thanks, we tested only this bacterial species (Lactobacillus casei NCAIM B 1147), as we are working for more species but this needs more time and cannot finish or publish in the current MS, thanks again

We are planning to use the following bacteria species as well:

- Bifidobacterium bifidum NCAIM B 02021

- Lactobacillus acidophilus NCAIM B 02085

- Lactobacillus delbrueckii subsp. bulgaricus

- Streptococcus thermophilus

3. Although it is stated in the future work, it would be nice to see a figure with some growth experiments or viability to see if these secreted nanoparticles are antimicrobial towards another bacterium.

Response: Many thanks, based on our previous studies on nano-Se and nano-Te,

We found that when we tested Lactobacillus sp., Bifidobacter sp., Streptococcus thermofilus and different kind of mixtures from various strains each bacteria strain produced nano-Se spheres in different size ranges as follows: Lactobacillus sp. :100- 200 nm, Bifidobacter sp. 400-500 nm, Streptococcus thermofilus: 50-100 nm. By the selection of bacteria used for production of high purity elemental red and grey selenium it was a very important factor to choose bacteria being already used and allowed to use in the food industry because our new product in this manner meets the strict quality requirements regarding for food supplements, additives. This technology seems to be more effective than the chemical synthesis, because it results relatively regular and uniform sized, high purity selenium spheres (100-500 nm, bacterium depending), production process is cheaper, faster and parameters can be controlled better. One of our future plans is to share this novel biological Nano-Se as food supplements, forage additives or plant nutrition. Summarizing the goals, our fundamental endeavor was to produce a non-toxic selenium form by the means of granted microbes, which are used in the food industry, without any non-desired side effects, enhancing resistivity toward illnesses, increasing the blood antioxidant level and boosting the quality and quantity of agricultural products as a fertilizer or forage supplement. According to the utilization we can differentiate two, nano-selenium containing products. First is the purified elemental nano-selenium (Nano-Se), that is mainly used for further laboratory and nanotechnological experiments, but later we intend to produce it for utilizing in different kind of medicinal supplements or fertilizer additives in industrial scale (Eszenyi et al. 2011).

4. Did the authors confirm if the secreted nanoparticles are toxic to lactobacilli?

Response: Many thanks

I have carefully considered your suggestion regarding the assessment of bacterial activity, anticancer properties, antibiofilm potential, and other relevant activities of the fabricated materials. I would like to clarify that the primary objective of the current manuscript was to elucidate the behavior of bacteria when exposed to selenite or tellurite within specific concentration ranges, particularly in terms of their differentiation capabilities in growth media.

However, I am delighted to inform you that I am actively preparing to address the broader spectrum of activities you mentioned in my upcoming research endeavors. I am currently in the process of designing experiments that will specifically investigate bacterial activity towards another bacterium and assess antibiofilm potential of the synthesized materials. I am optimistic that this forthcoming work will yield significant insights into these critical aspects, and I am committed to disseminating the outcomes through a subsequent publication.

5. How do the lactobacilli respond to the nanoparticles they secrete?

Response: Many thanks

Lactobacilli exhibits high tolerance to the nanoparticles it secretes. The Growth phase is seeming longer than control ones. Once it is reaching the late log phase, the pH of media increases more acidic where the synthesis of nanoparticles reaches maximum. As far as the cellular biomass is concerned, it is lesser than the control which confirms that the Lactobacilli has certain growth inhibition which may be due to the metal toxicity. Nanoparticle formation is usually considered as the defense mechanism against metal toxicity. It includes the enhanced excretion or reduced absorption of the metals as their primary response to exposure to high metal concentrations. It includes either reductive precipitation or volatilization of selenite/tellurite to Se⁰/ Te⁰/SeTe-NAs.

Minor issues

1. Sometimes the figures felt a little hard to follow. I suggest making the text and the figures on the same page if possible.

Response: Many thanks, we followed that as possible, thanks

2. The figure legends seem very brief and need a few more details to improve readability.

Response: Many thanks, all legend of figures were improved as possible, thanks

3. The EM images in figure 2 appear a bit blurred especially the middle one and if a better quality image could be provided this would be great.

Response: Many thanks, was changed to be better, thanks

Overall, a nice study with huge potential for the future.

Response: Many thanks, really, we appreciate again your great encouragements

Thanks a lot again!

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript was accepted because the author improved it in response to almost all of the suggestions. However, please include the text that explains nanoparticle formation from lines 235 to 273 in the manuscript and delete the sentences from lines 232 to 235.

Author Response

Reviewer 1#_Round 2

Comments and Suggestions for Authors

The authors would like to thanks for Reviewer 1 comments that improve our manuscript quality.

The manuscript was accepted because the author improved it in response to almost all of the suggestions. However, please include the text that explains nanoparticle formation from lines 235 to 273 in the manuscript and delete the sentences from lines 232 to 235.

Response: Many thanks for your comments.

• The sentences from lines 232 to 235 have been deleted and we merged the sentences from line 235 to 273 as the reviewer requested, thanks and for your comments and your time.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Author,

Thanks for re-submitting. I have not feedback regarding fig 6 and fig 7. I trust that the manuscript is now in an appropriate format.  

Author Response

Reviewer 2#_Round 2

Comments and Suggestions for Authors

The authors would like to thanks for Reviewer 2 comments that improve our manuscript quality.

Thanks for re-submitting. I have not feedback regarding fig 6 and fig 7. I trust that the manuscript is now in an appropriate format.  

Response: Many thanks for your time and comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

I can see the authors have responded to my initial comments and its clear that further work is planned to address some of these which is fair for the present manuscript. I have no further comments at this time. 

Author Response

Reviewer 3#_Round 2

Comments and Suggestions for Authors

The authors would like to thanks for Reviewer 3 comments that improve our manuscript quality.

I can see the authors have responded to my initial comments and its clear that further work is planned to address some of these which is fair for the present manuscript. I have no further comments at this time. 

Response: Many thanks for your time and comments.

Author Response File: Author Response.pdf