Optimization of Production of Polyhydroxyalkanoates (PHAs) from Newly Isolated Ensifer sp. Strain HD34 by Response Surface Methodology
Round 1
Reviewer 1 Report
Reviewer comments:
The presentation of this manuscript was good enough in terms of structure but some improvement of English language in needed. Moreover, the paper is subjected to major improvement.
1. Author did all the experiments in flask without controlling pH, then how can the author sure that pH 9 is the optimal. Did the author measured the final pH and how they are differ from initial pH.
2. Effect of production medium was described in Materials and Methods but the effect is not clear to me even not discussed properly in results and discussion parts.
3. The substrate and medium was used in evaluation of the pH, temperature, inoculum ……….. need to mention.
4. Shake flask experiment for more than 100 h was too long time and the process conditions (pH, DO) was changed a lot during the process that also effect the physiological behavior of the organism that ultimately effect the optimum condition determined through this study.
5. The maximum temperature 450 C for DSC was too high temperature to evaluate the polymer properties through DSC.
6. Page 5 line 236: ‘The result was compared with the earlier reports’ – is not a part of material and methods. There were a number of similar sentences, need to rewrite.
7. Please subdivided the optimization of PHAs production results (3.4). It was completely mixed up, made complex for readers. Separate clearly the effect of pH, temperature, production medium and others………..
8. The meaning of the superscripts (a,b,c,d) used in Table 5 was not clear.
9. The meaning of the marks (a,b,c,g,ab,bc…………) used within the Figure 5 need to explain.
10. Reason behind the decreasing of PHA yield with inoculum size was not clear.
11. Provide more clear picture.
12. Figure 7 was missing.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments:
This manuscript titled “Optimization of polyhydroxyalkanoates (PHAs) production 2 from newly isolated Ensifer sp. strain HD34 by response sur- 3 face methodology.” deals with the the production and medium optimization of polyhydroxyalkanoates (PHAs). The authors claim that among 341 isolates from 40 composted soil samples, the best isolate was HD34 strain which was identified using morphological, molecular, and bio-chemical methods. The authors further claims that the result showed that the strain was the most closely related to Ensifer adhaerens 19 LMG20216T with 99.6% .The work has been presented systematically and the results are thoroughly explained on the basis of obtained results. I feel that the manuscript may be accepted for publication after minor revision taking into account below points:
Comment: 1
There exist some errors in this article, both typo and grammatical which has reduced the quality of the manuscript to a greater extent. Moreover, expressions of some sentences are not well enough in aspects of the professionalization and accuracy. Therefore, it is important to carefully read and further modify the entire manuscript and check grammatical errors.
Comment: 2
It is recommended for the authors to provide the schematic representation of the preparation of the polyhydroxyalkanoates (PHAs) production for better understanding for the readers.
Comment: 3
The introduction part is not appropriate and much of the work based on synthesis of nanocomposites have not been cited and discussed. Author must cite some latest relevant papers such as: Polymers 2022, 14(11), 2224; https://doi.org/10.3390/polym14112224; Bio-plastic Polyhydroxyalkanoate (PHA): Applications in Modern Medicine. In Bioplastics for Sustainable Development (pp. 231-257). Springer, Singapore.; Applied microbiology and biotechnology 104, no. 7 (2020): 3121-3131.; Boron nitride doped polyhydroxyalkanoate/chitosan nanocomposite for antibacterial and biological applications." Nanomaterials 9, no. 4 (2019): 645.
Comment: 4
The author must explain about their contribution in the last part of their introduction more appropriately.
Comment: 5
The methods sections are not appropriately written. The author must discuss in detail the amount of precursors material. The author must write the details so that if anyone wants to perform the experiment, he must have appropriate data to perform the synthesis.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript by Khamkong et al. is very well conceptualized and presented. Authors gave a good insight in problematics and performed impressive number of various experiments. Very detailed and well-designed study. Results are thoroughly discussed and connected with previous research. However, language and style need to be improved, so here are few suggestions/corrections from my point of view:
T1. The first time an abbreviation is used, it is important to spell out the full term and put the abbreviation in parentheses.
22. The strain was identified using morphological, molecular, and biochemical methods, so it should be plural – results.
L19: The result showed that the strain was the most closely related to Ensifer adhaerens…
L26: The result…
33. It could be: “complete biodegradation” or “the ability to completely degrade by various mo”
L37: The interesting points of PHAs are completely biodegradability by…
44. This is not quite true. Quoted manuscript does not state this. It might be better to say: “with the properties that are similar to the petroleum polymers polypropylene (PP) and polyethylene (PE)”
L39: …and properties same as polypropylene (PP) and polyethylene
55. “Gram-negative bacteria are typically used for production of PHAs on the industrial scale.”
L46: On the industrial scale, PHAs production has been typically used Gram-negative bacteria.
66. I am guessing these two sentences should be combined in one?
L60: Because of biocompatibility and biodegradability. The PHAs family has been interested in applying to industrial, medical, pharmaceutical, and agricultural fields.
77. It should be: “The soil samples”
L78: The sample soils…
88. “After 7 days of cultivation”
L82: After 7 days of culture..
99. Plural: “conditions”
L83: …and was shaken at the same condition
110. Authors should also mention which method was used for phylogenetic tree, outgroup microorganism they have used and bootstrap test if performed as well. So basically, their Figure 3 caption.
L126: …and a phylogenetic tree was created using MEGA X software.
111. Authors should uniform writing numbers. Every number higher than 10 should be written as a number. So it should be 50, not fifty.
112. Authors are presenting multiple conditions, so it should be plural: “conditions were”. Authors should check the manuscript for similar mistakes.
L184: The incubation condition was…
113. “were selected”
L194: … was selected
114. Authors mentioned use of scanning electron microscope for strains’ characterization, and they reported SEM micrograph in Figure 2. Nevertheless, Materials and Methods are missing details on performed SEM analysis.
115. Table caption needs to be rewritten. It could be: “Amount of accumulated PHAs granules per cell and overall PHAs content in positive isolates”
L277: Fifteen positive isolates which could accumulated PHAs and their effective on PHAs pro- duction
116. “After the experiment under optimal conditions was performed the effects of incubation time, pH, inoculum size, and temperature were investigated.”
L391: After the optimal medium was performed, including PDB medium and glucose…
117. “Longer incubation time”
L393: more incubation time affected more cells growth of Ensifer sp. HD34
118. Authors should use terms such as “using larger inoculum” or “increased inoculum”
L419: …using higher inoculum size
119. “Rapid”
L423: …might cause rapidly nutrient consumption
Author Response
Please see the attachment
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Authors try to incorporate all the issues that raised during the 1st review. My opinion is positive on this manuscript.
Author Response
We would like to thank the reviewer for your thoughtful comments and efforts towards improving our manuscript, as well as the important guiding
significance to our researches.
