Biosynthesis of Polyhydroalkanoates Doped with Silver Nanoparticles Using Pseudomonas putida and Pseudomonas aeruginosa for Antibacterial Polymer Applications

No

Comment / response

C1

Line 123: typos in "polydydroxybutyric acid" and "polyhydroxybutyrate":

R1

Author response: Thank you very much for your observation, the corresponding corrections were made and are indicated in the manuscript.

C2

Please discuss a peak at around 450C on TGA curve (Fig.3B) it indicate presence of entity with different thermal properties.

R2

Thank you very much for your comment. A peak at around 450°C on a thermogravimetric analysis (TGA) curve of polyhydroxybutyrate (PHB) could also be attributed to impurities possibly introduced from the carbon source during the synthesis, processing, or handling of PHB. These impurities, including organic contaminants with higher thermal stability than PHB, might degrade at higher temperatures. Unfortunately, due to time constraints, it was not possible to include tests to evaluate the purity of the obtained materials.

C3

In discussion of FTIR, which is executed correctly, please indicate the form of modification of PHAs with Ag NPs - are they covalently bound or rather physically?

R3

A reduction in the intensity of peaks corresponding to functional groups involved in binding hydroxyl or carbonyl groups in C-H stretching, shifts in the C-H stretching region (2800-3000 cm⁻¹) if there is modification of the hydrocarbon chain, and changes in the carbonyl stretching frequency (around 1700 cm⁻¹) might indicate covalent attachment of AgNPs. PHAs have carbonyl groups that could interact with silver, leading to the possible formation of silver-oxygen bonds (Ag-O) if AgNPs interact with ester or hydroxyl groups in PHAs. This explanation was included in the new version of the manuscript.

C4

The legend and scale on Fig.7 and Fig.8 are unreadable - please correct.

R4

Thank you for your observation; the figures have been corrected.

C5

In line 370 Authors mention XRD as the technique of physico-chemical analysis while no other information in the manuscript can be found on adapting XRD.

R5

The XRD analysis is included in the supplementary information. The XRD spectrum of PHAs-Gly exhibited broad bands around 2θ = 20.10°, 30.24°, and 39.91° (Figure S1). The peak near 20.10°, as well as the hump at 30.26°, can be attributed to the XRD pattern of poly(3-hydroxybutyrate) (P(3HB)) according to the literature (Frone et al. 2020). On the other hand, the shifted signals may be due to some residual moisture in the material, while the broad bands are likely due to the contribution of both the crystalline peak of mcl-PHA and the amorphous phase of P(3HB) described in the literature (Sedlacek et al., 2020).

Figure S1. X-ray diffraction pattern of PHAs obtained from P. aeruginosa strain and reagent-grade glycerol.

References:

·  Frone, A.,N., Nicolae, C.,A., Eremia, M.,C., Tofan, V., Ghiurea, M., Chiulan, I., Radu, E., Damian, C.,M., Panaitescu, D.,M., Low molecular weight and polymeric modifiers as toughening agents in poly (3-hydroxybutyrate) films. Polymers 2020, 12 2446.

·  Sedlacek, P., Pernicova, I., Novackova, I., Kourilova, X., Kalina, M., Kovalcik, A., Koller, M., Nebesarova, J., Krzyzanek, V., Hrubanova, K., Masilko, J., Slaninova, E., Trudicova, M., & Obruca, S. Introducing the Newly Isolated Bacterium Aneurinibacillus sp. H1 as an Auspicious Thermophilic Producer of Various Polyhydroxyalkanoates (PHA) Copolymers–2. Material Study on the Produced Copolymers. Polymers (Basel) 2020, 12 1298.

No

Comment / response

C1

Title is not clear. “Biosynthesis of polyhrdroxyalkanoates from crude glycerol using Pseudomonas species and doping with silver nanoparticles for antibacterial applications”

R1

Thank you very much for your observation, The title of the article was modified by “Biosynthesis of polyhydroalkanoates doping with silver nanoparticles using Pseudomonas putida and Pseudomonas aeruginosa for antibacterial polymers applications”

C2

Abstract should be re-written. First sentence states that “PHAs extracted from Pseudomonas strains” which is contrary to title and second sentence in abstract. Abstract should also state the results observed by including the statistical data, but not by briefly stating the observed results.

R2

Thank you very much for your observation, the summary was modified.

These PHAs were produced using reagent grade glycerol and crude glycerol as the carbon source. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production could be obtained using Pseudomonas putida compared to Pseudomonas aeruginosa; and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the Pseudomonas aeruginosa strain.

C3

Introduction should state the novelty and the objectives of the work clearly at the end.

R3

The corresponding correction was made and is presented in the new version of the manuscript.

C4

Line 104: What is the basis for choosing “0.5 ml” of glycerol in both cases? In Figure 2, concentration of glycerol and crude glycerol were indicated as “g/L”, whereas quantity is mentioned as “ml” in section 2.1. (Line 104).

R4

Thank you very much for your comment. You are right, there was an-error which has already been corrected, 0.63 g/L was used. Higher initial cell densities can lead to the rapid accumulation of inhibitory products and the early depletion of essential nutrients. Maintaining a low initial cell density ensures that substrates, such as glycerol, are used more efficiently during exponential cell growth. This is crucial to avoid wasting substrates and to maximize PHA production before resources become limiting. Optimal growth and PHA production are reported with the use of carbon source substrates in the range of 0.1 to 1 g.

References:

Ronďošová, S., Legerská B., Chmelová, D., Ondrejovič, M., Miertuš, S., Optimization of Growth Conditions to Enhance PHA Production by Cupriavidus necátor, Fermentatios, 2022, 8 451.

Lutz lenczak, J., Schmidell W., Falcão de Aragão G., High-cell-density culture strategies for polyhydroxyalkanoate production: a review. JIMB, 2013 40, 276-286.

C5

Figure 5 and 6 titles are confusing. Why there is change in wavenumber range for regent grade glycerol and crude glycerol? In general, FTIR spectra will be shown from 4000  to 500 range, but that is not the case here. Figure 5 and 6 titles are confusing. Why there is change in wavenumber range for regent grade glycerol and crude glycerol? In general, FTIR spectra will be shown from 4000  to 500 range, but that is not the case here.

R5

Thank you very much for your comment. You are right, figures 5 and 6 are confusing, which is why they were modified for better understanding and included in the manuscript.

C6

Section 2.6. states about nanocomposite of PHA-AgNPs, but Figure 7 and 8 indicates films, is the author objective is to synthesize nanocomposite or film? Section 3.5. Line  320: states Figure 7 and 8 are for crude glycerol micrographs, but Figure titles say “reagent grade glycerol”. SEM images should be of same scale. Figure 7 and 8 sub figures should be clearly indicated.

R6

The objective was to evaluate the antimicrobial activity of PHAs doped with silver nanoparticles, without employing any technique for film production. Antibacterial disk tests were conducted with the different nanocomposites obtained through the sonication of PHAs and silver nanoparticles at two distinct concentrations: 0.05 mg/kg and 0.5 mg/kg. For these tests, filter paper disks were impregnated with the various nanocomposites, PHAs obtained from crude glycerol, and reagent-grade glycerol at different concentrations, in order to evaluate their antibacterial performance. Images 7 and 8 were modified and suggested corrections were made.

C7

7. Methodology for antibacterial activity? Should be indicated as “antibacterial” activity in manuscript, not as “antimicrobial” activity as only one bacterium was used for study. S. epidermis or S. aureus? Which bacteria was used? No inhibitory zones were observed in Figure 10 unlike Figure 9. Tabulate the inhibitory zone values in mm.

R7

Thank you very much for the observation, the term "antimicrobial activity" was replaced by "antibacterial activity" and it was specified that the bacteria used was Staphylococcus aureus. The suggested table was added to the manuscript.

C8

8. Only 6 out of 51 references are recent (on or after 2020). Should include recent references.

Thank you for the observation, the modification was made by replacing some with more updated references.