Cocoa Bean Shell as Promising Feedstock for the Production of Poly(3-hydroxybutyrate) (PHB)
Round 1
Reviewer 1 Report
The manuscript by Sánchez and co-authors reports the use of cocoa bean shells as a promising substrate for P(3HB) production. See some comments below:
General comments
- The correct terminology is Poly(3-hydroxybutyrate). Please correct the entire text. Please attain to IUPAC standard terminology https://www.ebi.ac.uk/chebi/searchId.do?chebiId=53389
- Could you estimate a percentage of cost reduction in the PHB production process using CBS? Can you provide CBS price and calculate the final PHB production cost from this byproduct?
Specific comments
L 12. Is 107 mg PHB/g dry matter the yield? If so, use this terminology.
L 40-41. Which carbohydrates? Pentoses, hexoses?
L 48-49. PHA is also a source of reducing power .
L 52-53. Correct all scientific nomenclature to italics: "Bacillus, Azotobacter, Pseudomonas, and Rhizobium". Correct the entire text.
L 76. H2SO4. Same for L 135, 155.
L 89-90. It is not clear whether you supplemented the obtained hydrolysate with typical culture media components, as nitrogen, magnesium, calcium, iron etc. Please clarify.
L 98-103. In the case of the medium without solids, could you follow cell growth by optical density or weighting the cell dry mass?
L 105. It is not clear why this procedure was performed. Please clarify.
L 127-128. The last sentence should be moved to the results section.
L 137. Other typical hemicellulosic carbohydrates can be present in your hydrolysate, as xylose, arabinose etc. Why only glucose was measured?
L 143. Other typical hemicellulosic carbohydrates can be present in your hydrolysate, as xylose, arabinose etc. Why only glucose was measured?
L 160-161. As you have access to an HPLC, you could have used this to measure the carbohydrates in you hydrolysate - glucose, xylose, arabinose etc. Is there a reason why you've used a not so accurate methodology for carbohydrates determination?
L 172. Define "P".
L 176. Are the bacterial cells included in the "total suspended solids"?
L 176. Is your bioprocess a real fermentation, i.e. anaerobic?
L 190. Why did the soluble reducing sugars concentration decreased in the centrifuged medium? If they are soluble the concentration should have not varied. Same question for soluble phosphorus concentration.
L 213-215. Which condition was achieved for PHB accumulation in your experimental setup? Which was the limiting nutrient?
L 226-227. Some strains have been demonstrated to tolerate acetate and HMF present in lignocellulosic hydrolysates, please check doi.org/10.1016/j.indcrop.2020.112703 and https://doi.org/10.1007/s10295-014-1485-5
L 351. Figure 3 - really hard to interpret PHB concentration over time in this figure. I suggest including this information in a new graphic or table.
L 354-356. As mentioned above, not clear from figure 3. Please include a new figure and rewrite this part of the text accordingly.
L 368-379. As you did not observe a significant increase in bacterial biomass, what are your sugars being used for?
L 384-386. Some suggestion as for figure 3, include a new figure with PHB concentration over time. Include also a volumetric productivity over time g/L.h in this graphic.
L 392-394. Do you have more information for the tested strain accumulating PHB during growth in different culture media and/or different carbon sources?
L 402-403. C. necator sugar consumption range is very limited when compared to other platform strains (check doi.org/10.1016/j.biortech.2021.125472). Do you have genome information for B. firmus? This would be really useful to determine the present catabolic pathways, and also to propose metabolic engineering studies.
L 455-456. Define the used interval (maybe show in your graphic).
L 488-490. Was 3HB the only detected monomer? Please include a chromatogram in the supplementary material indicating internal standard and 3HB peaks.
L 538-540. Could the solids present in the culture media represent an additional obstacle for PHB purification from bacterial cells?
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
1- What is the suggestion to increase the PHB production from CBS?
2- Is it possible to produce PHA from CBS?
3- What is the length of the carbon chain in the structure of PHB?
4- After how many days the PHB from CBS will degrade? Is the degradation duration the same in soil and water?
5- What are the other by-products similar to CBS to produce the same amount of PHB?
6- As a preliminary study, it is suggested to use GC to find out the components of the CBS.
7- The authors can improve the introduction using the following references:
Sabbagh, F., & Muhamad, I. I. (2017). Production of poly-hydroxyalkanoate as secondary metabolite with main focus on sustainable energy. Renewable and Sustainable Energy Reviews, 72, 95-104.
Mojaveryazdia, F. S., Zainb, N. A. B. M., & Rezaniac, S. (2013). Production of biodegradable polymers (PHA) through low cost carbon sources: Green Chemistry. International Journal, 4(3).
Siddiqui, M. W., & Bansal, V. (2017). Polyhydroxyalkanoates: A valuable secondary metabolite produced in microorganisms and plants. In Plant Secondary Metabolites, Volume Three (pp. 205-234). Apple Academic Press.
Mojaveryazdi, F. S., Muhamad, I. I., Rezania, S., & Benham, H. (2014). Importance of glucose and pseudomonas in producing degradable plastics. Jurnal Teknologi, 69(5).
8- There are some typo errors in the manuscript that must be edited by an EN service.
9- Just as a suggestion, add some future prospects to the conclusion.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Please consider using "cultivation" and not fermentation to describe process. Fermentation is classically used for physiological process in which ethanol or lactic acid are produced - did you detect any of these compounds in your cultivation?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
