Release and Purification of Poly(3-hydroxybutyrate) P(3HB) via the Combined Use of an Autolytic Strain of Azotobacter vinelandii OP-PhbP3⁺ and Non-Halogenated Solvents

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study reported the scale up fermentation of PHB by a previously constructed engineered strain wihich could undergo self-lysis during fermentation. In general, the novelty is lacked here in this manuscript becourse the merely application of new solvent which inproved the total extraction yield for several percent can not be called "novel study", except the proposed process optimization achieved siginificant improvement comparing with previous studies. In addition, several sections demand significant revision, as listed in detailed comments.

(1) low quality of figure 4. what information can be seen in black box???

(2) if cells undergo lysis during fermentation, why protein level decrased?? In my point of view, aquesous protein level should be increased from broken cells. 

(3) lack in depth discussion of the optimization with other studies. The only difference is the solvent applied. Could the proposed method be applied in industrilization to decrease the high cost during purification of PHB? Comparing with traditional strain (no lysis) and solvent, how much cost can be reduced? these information may atrtact interests from readers, if the major topic of this manuscript covers the process optimization.

(4) for a full long research artical, more detailed experimental data should be supplemented, e.g. Mw by GPC, qualitative and quantitative assay of impurities in final products and et. al. 

Author Response

Release and purification of Poly-3-hydroxybutyrate P(3HB) by the combined use of an autolytic strain of Azotobacter vinelandii OP-PhbP3+ and non-halogenated solvents

 

Reviewer 1:

The study reported the scale up fermentation of PHB by a previously constructed engineered strain which could undergo self-lysis during fermentation. In general, the novelty is lacked here in this manuscript because the merely application of new solvent which inproved the total extraction yield for several percent cannot be called "novel study", except the proposed process optimization achieved significant improvement comparing with previous studies. In addition, several sections demand significant revision, as listed in detailed comments.

 

• Low quality of figure 4. what information can be seen in black box???

          R: Thank you for your comments. Figure 4 shows the cells before lysis with intracellular granules and shows the cells with the granules released into the medium. In the revised version of the manuscript, the quality of the figure has been improved.

• If cells undergo lysis during fermentation, why protein level decrased?? In my point of view, aquesous protein level should be increased from broken cells.

    R: Thank you very much for the reviewer's comment. Since the protein determination is performed on the cell pellet, which is separated after centrifugation of the medium, the measured protein content decreases in the pellet. Consequently, a decrease in protein concentration within the insoluble fraction after a cell lysis event is consistent with the assumption that a significant portion of the proteins is released into the soluble fraction following cellular disruption.

This paragraph was included in the new version of the manuscript (page 10, lines 529-531).

3) Lack in depth discussion of the optimization with other studies. The only difference is the solvent applied. Could the proposed method be applied in industrialization to decrease the high cost during purification of PHB? Comparing with traditional strain (no lysis) and solvent, how much cost can be reduced? this information may attract interests from readers, if the major topic of this manuscript covers the process optimization.

     R: Thanks for the comment. The present method presents a clear potential for large-scale industrial implementation, as it combines high extraction and purification efficiency with a markedly low environmental footprint. One of its main advantages lies on the use of CYC solvent, which not only exhibits a strong capacity to solubilize the polymer, but in addition can be efficiently recovered by distillation and reintroduced into subsequent extraction cycles.

 

All the above, reduces solvent consumption and, consequently, decreases the operational costs associated with raw materials, making the process more sustainable and economically attractive. Another critical factor is the biological system employed. The strain used is a well-established PHB overproducer that, unlike other commonly studied strains such as the OP strain, naturally releases polymer granules into the culture supernatant. This intrinsic characteristic eliminates the need for energy-intensive cell disruption steps and simplifies downstream processing, thereby facilitating polymer recovery and ensuring high purity levels. The ability to obtain extracellular granules not only reduces the complexity of the purification stage but also minimizes the use of additional chemicals, further aligning the process with environmentally responsible practices.

 

In this context, the combination of an efficient solvent recovery strategy with a biologically optimized strain provides a platform that significantly improves the cost-effectiveness of biopolymer production. These advantages highlight the feasibility of scaling up this method to an industrial level, where the reduction in separation and purification costs can have a determinant impact on the competitiveness of P3HB and related bioplastics as sustainable alternatives to petroleum-derived plastics.

 

In the new version of the manuscript these paragraphs were included (page 16, lines 816-835).

 

• For a full long research article, more detailed experimental data should be supplemented, e.g. Mw by GPC, qualitative and quantitative assay of impurities in final products and et. al.

 

R: We sincerely appreciate the reviewer’s valuable suggestion. In the present manuscript, our primary objective was to demonstrate the feasibility and efficiency of the proposed method. In addition, data on the molecular weight of the product obtained using the proposed method (determined by GPC), as well as its corresponding analysis, have been included (Figure 7).  However, we fully agree that the inclusion of more detailed experimental data, such as the FTIR analysis to identify and characterize possible impurities in the polymer would be highly desirable in future studies.

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

 

I reviewed your intriguing manuscript, which presents a novel process for P(3HB) biopolyester production by and recovery from an Azotobacter vinelandii strain overexpressing the phasin PhbP3. It was shown that high intracellular P(3HB) fractions were obtained, and that the strain undergoes autolysis after a certain cultivation time, which in time contributes to a convenient downstream processing. Two treatment methods for biomass and recovered P(3HB) were evaluated: SDS treatment and extraction using the non-halogenated solvent cyclohexanone. Especially the latter resulted in high recovery yields and high polymer purity. It is suggested that the combination of a phasin-overexpressing production strain and an environmentally benign solvent contributes to make P(3HB) more efficient and sustainable.

The work is of scientific interest, and contributed to current endeavors to optimize the downstream processing as one of the crucial stages during the PHA production process. The work is unique, and the experiments are well planned and reproducible for peers.

Some aspects should be addressed in order to further improve the manuscript:

1. Unfortunately, line numbering is missing, which makes it cumbersome to comment the text.
2. Be consistent with polymer names and their abbreviations: you mix “poly-3-hydroxybutyrate” and “polyhydroxybutyrate”; it should be “poly(3-hydroxybutyrate)” (also in title!). you mix PHB, P3HB, and P(3HB). I suggest using P(3HB) consistently.
3. Abstract, page 2, page 3, etc.: “molecular weight“: which?? Weight average molecular weight? Mass average molecular weight?
4. Abstract: 2 (respect to PHB initial P(3HB)”: unclear!
5. Page 2: “as short-chain and medium-chain PHAs”: what about long-chain PHAs?
6. Page 2: “such as batch and fed-batch”: what about continuous processes for PHA production? Suggested to refer to a standard article published on this topic in Fermentation: Koller, M. (2018). A review on established and emerging fermentation schemes for microbial production of polyhydroxyalkanoate (PHA) biopolyesters. Fermentation, 4(2), 30.
7. Page 2: “products of lipid nature“: “lipophilic products”
8. Page 3: References [15-20]: not all of them refer to „bacteriophages that infect bacteria”, e.g., [17] uses the genetic info for lysozyme production from Gallus gallus.
9. Page 3: “6.0 ± 0.04 mmol L⁻¹ h-1“: „-1“ in superscript
10. 3: “Holland”: should be “The Netherlands” (Holland is only a part thereof)
11. 3: explain “Di/DT“
12. 4.2: „of H2SO4 at 90 °C“: concentration of acid?
13. 4.3: “Mean molecular mass“: thsi terminus is unclear: should be average molecular mass (weight or number; see above comment)
14. 6: there is a comment text field to be removed
15. 6: what are “independent cultures”?
16. Generally: “et al”: “et al.»

Author Response

Reviewer 2:

I reviewed your intriguing manuscript, which presents a novel process for P(3HB) biopolyester production by and recovery from an Azotobacter vinelandii strain overexpressing the phasin PhbP3. It was shown that high intracellular P(3HB) fractions were obtained, and that the strain undergoes autolysis after a certain cultivation time, which in time contributes to a convenient downstream processing. Two treatment methods for biomass and recovered P(3HB) were evaluated: SDS treatment and extraction using the non-halogenated solvent cyclohexanone.

 Especially the latter resulted in high recovery yields and high polymer purity. It is suggested that the combination of a phasin-overexpressing production strain and an environmentally benign solvent contributes to make P(3HB) more efficient and sustainable.

 

The work is of scientific interest and contributed to current endeavors to optimize the downstream processing as one of the crucial stages during the PHA production process. The work is unique, and the experiments are well planned and reproducible for peers.

 Some aspects should be addressed in order to further improve the manuscript:

• Unfortunately, line numbering is missing, which makes it cumbersome to comment the text.

R: Thank you for the remark. We will include the number of lines in the revised version.

 

• Be consistent with polymer names and their abbreviations: you mix “poly-3-hydroxybutyrate” and “polyhydroxybutyrate”; it should be “poly(3-hydroxybutyrate)” (also in title!). you mix PHB, P3HB, and P(3HB). I suggest using P(3HB) consistently.

 

R: Thank you for the remark. We have standardized the name to poly(3-hydroxybutyrate) and used the abbreviation P(3HB) consistently throughout the manuscript.

 

• Abstract, page 2, page 3, etc.: “molecular weight“: which?? Weight average molecular weight? Mass average molecular weight?

 

R: Thank for the comment.  In the new version of the manuscript we included in all cases “weight average molecular weight”.

 

• Abstract: 2 (respect to PHB initial P(3HB)”: unclear!

R: Thank you for the comment. We have clarified that paragraph in the text, indicating that we are referring to the yield and purity of P3HB extracted from the cellular biomass (page, 1 lines 23-26).

 

• Page 2: “as short-chain and medium-chain PHAs”: what about long-chain PHAs?

 

R: Thank you for the remark. In the new version, we included “short-chain, medium-chain, or long-chain PHAs” (page 2,  lines 82-83).

 

• Page 2: “such as batch and fed-batch”: what about continuous processes for PHA production? Suggested to refer to a standard article published on this topic in Fermentation: Koller, M. (2018). A review on established and emerging fermentation schemes for microbial production of polyhydroxyalkanoate (PHA) biopolyesters. Fermentation, 4(2), 30.

R: Thanks for the suggestion, in the new version of the manuscript we have included the indicated reference (page 2,  line 91).

 

• Page 2: “products of lipid nature“: “lipophilic products”

R: Thanks for the comment, we have already included the modification in the text (page 2,  line 99).

 

• Page 3: References [15-20]: not all of them refer to „bacteriophages that infect bacteria”, e.g., [17] uses the genetic info for lysozyme production from Gallus gallus.

R: Thanks for the comment. We made the correction in the new version (page 3, line 183).

 

• Page 3: “6.0 ± 0.04 mmol L⁻¹ h-1“: „-1“ in superscript

R: Thanks for the comment, we have already included the modification in the text (page 3, line 189).

 

• Page 3: “Holland”: should be “The Netherlands” (Holland is only a part thereof)

R: Thanks for the comment, we have already included “The Netherlands” in the text (page 4, line 288).

 

• explain “Di/DT“

R: Thanks for the comment. Di/DT is the ratio between the diameter of Rushton impeller for a bioreactor 3 litters on diameter tank of 3 litters. This was included in the new version (page 4, lines 290-291).

 

• 2: „of H2SO4 at 90 °C“: concentration of acid?

           R: Thank you for the observation. We have now specified “concentrated H₂SO₄,” which is typically ~98%.

 

• 3: “Mean molecular mass“: thsi terminus is unclear: should be average molecular mass (weight or number; see above comment).

    R: Thank you so much for the comment. In this study, we used the term average molecular mass weight. For clarification, the term "mean molecular mass" was changed to "average molecular mass weight" throughout the article.

 

• 6: there is a comment text field to be removed

R: Thank you for the observation. We have removed the comment text.

 

• 6: what are “independent cultures”?

R: Independent cultivations were defined as bioreactor kinetics performed at separate times using independent inocula, under identical operating conditions, and conducted in triplicate.

 

 

• Generally: “et al”: “et al.»

R: Thank you for the observation. All references have now been corrected to “et al.”

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript present improved quality, although its novelty is still lacked.