Entrapping Immobilisation of Lipase on Biocomposite Hydrogels toward for Biodiesel Production from Waste Frying Acid Oil

Round 1

Reviewer 1 Report

-English style and grammar must be revised. Extensive editing of English language and style is necessary.

The paper presents several issues and should be improved:

- The authors presented water content and swelling behaviour but no correlation with catalytic results was presented. My question is: what is the interest of hydrogels characterisation for the paper?

- The authors said: “Therefore, enzyme conformation showed more stable. Since, it was not destroy by both physical force and chemical reaction.” Can you explain this statement? Please support your evidences with literature.

- lines 180-185: I can’t understand these sentences you must re-write it.

- line 215: “According to the highest biodiesel yield of GAL (75.00%) 2.66 mg KOH/g oil and PAL (77.83%) 2.36 mg KOH/g oil were achieved at 50°C.”

What is the acid number of the initial FFA+WFAO mixture? How do you calculate the biodiesel yield?

- line 390: “Free fatty acids (FFA) in WFAO (50 mL), GAL/PAL (2% w/v) and methanol were added together in 250 mL Erlenmeyer flask on incubator shaker to perform esterification reaction.”

Do you know the amount of FFA in the WFAO mixture?

- line 256: “Enzyme reusability is used to indicate how good potential for a biocatalyst”, please provide an explanation.

- line 316: “These parameters were estimated using regression models following to previous work [52]. It was interesting to note that the predicted biodiesel properties obtained from WFAO met the European biodiesel standard.” We should explain better the methodology used.

- line 427: You should correct the equation 5.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors

according to the second round of review and your answers to my comments. I am satisfied with their explanations and corrections. I recommend the manuscript in this form for further publication steps.
Best regards

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I this revised form I recommend the manuscript for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The paper reports the “Entrapping immobilisation of lipase on biocomposite hydrogels toward for biodiesel production from waste frying acid oil”. The topic is interesting although the paper presents several issued and should be improved.

 

- The introduction is very extensive and should be revised. Paragraph 3 is irrelevant so should be shortens. Last phrase of paragraph 6 should be re-written. “Recently, there was no one…”

- Why the authors presented the water content of hydrogels and swelling behaviour if they didn’t compared this with the lipase immobilised hydrogel which is the active catalyst for biodiesel production. So, characterisation of the immobilised lipase hydrogels should be also included to characterization section.

- Figures are not well referenced in the text.

- Discussion in section 2.2 is confusing and should be improved. Table 1 compares immobilisation yields of different immobilisation techniques although no comparison was made with same entrapment technologies performed by other authors. Please include this and explain why the entrapment is the best methodology comparing with others.

- The discussion of the results should be descriptive and not just a presentation of the results. It must contain an initial introduction of the subject before the presentation of results and this was not presented in this article. The discussion must be revised and corrected accordingly to suggestion.

- In section 2.3-2.7 authors should indicate in figures the experimental conditions used in each section.

- Please explain better Figure 8 and the importance of residual activity in biodiesel production.

- The authors presented Table 2 to compare the biodiesel production but used different processes, this study is a esterification processes and the others are transesterification processes. This is not comparative please provide similar processes to compare.

- English changes are required in all text.

In general the paper has too much information and is not properly presented or discussed.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Overall comments:

1. The manuscript is poorly written. The introduction paragraph must be improved in case of some unclear sentences and plenty stylistic errors.
2. The main aim of the study is not clearly defined. The purpose and significance of the research should be more emphasized.
3. The authors are asked to explain if the method proposed by them is new or some other research groups have been working on this approach before?
4. The materials and methods section should be improved and supplemented with a number of detailed information.
5. Results and discussion section should be carefully analyzed once more time by the Authors (the text requires major correction of linguistic mistakes).
6. The Conclusions section is too general. There is no information about the results achieved by the Authors at all. It should be improved with the comparison of the results with current literature reports, that could allow to prove the significance of performed study.
7. The whole manuscript contains many linguistic and grammatical errors (due to large amount of these mistakes I did not mark them in detailed remarks). Extensive editing of English language and style is required.

 

Detailed remarks to the manuscript

1. Lines 49 – Authors write about ‘hydrophobic support’ but their study concerns hydrogel materials (alginate, pectin, gelatin) that are hydrophilic in their nature. Please explain the reason why You write about hydrophobic carrier.
2. Lines 59-60 – ‘In addition, immobilised enzyme on hydrogels provides many advantages such as swelling, …’ – I would like to ask Authors to explain in what context You think that swelling is the advantage of the immobilisation carrier.
3. Line 65 – ‘…entrapment enzyme within alginate…’ – change the word ‘enzyme’ to ‘lipase’
4. Line 78 – ‘potassium ions’ – please reject this from the text because K⁺ is not divalent ion.
5. Lines 80-81 – ‘Gelatin based hydrogel was also used to entrap invertase from yeast cell wall for hydrolysis of sucrose. After 70 consecutive cycles, it retained 75% of its original activity’ – This is the information about completely different enzyme that is not the subject of presented manuscript. Authors are asked to find the example of lipase immobilised using gelatin hydrogel as the carrier.
6. Line 103 – ‘PA bead’ - Authors are asked to explain if only one bead of hydrogel was always used in each experiment. This remark applies to the entire manuscript.
7. Line 103 and 104 – 14%, 96,13% - I would like to emphasize that the difference between these values is negligible(within the limits of measurement error).
8. Line 109 – (See Fig.2) – I suppose that Authors meant the Fig.1.
9. Line 110 – The swelling process is varied and depends very much on the chemical composition of the tested hydrogel. The authors are asked to provide the chemical composition of the quoted work [24].
10. Line 119 – As shown in Fig.3 – I suppose that Authors meant the Fig.2.
11. Lines 119-120 – ‘As shown … to 80 °C’ – This sentence is unclear. Authors are asked to rephrase it.
12. Line 132 – ‘…hydrophobic support…’ To the best of my knowledge, unmodified hydrogel materials based on alginate, gelatin or pectin are hydrophilic in nature. Authors are asked to explain what kind of hydrophobic support they meant.
13. Line 152 – Please give the information if the immobilisation yield was determined in the same way (based on the protein content) in all papers cited in Table 1)?
14. Line 175 – ‘…in the right hand…’ – The Authors have used the colloquial statement and are asked to replace it with a more scientific term.
15. Lines 178-182 – ‘The highest biodiesel yield ……….and the lowest FFA (3.7310 mg KOH/g oil.’ – I do not agree with this statement. Based on the results presented in Fig.4 could be observed that the highest biodiesel yield and the lowest FFA were obtained rather at 5:1 methanol to fatty acids molar ratio for GAL and 7:1 for PAL respectively. Authors are asked to analyze the results one more time. Explanation is needed.
16. Line 167 (Fig.3), 193 (Fig.4), 215 (Fig.5), 232 (Fig.6), 246 (Fig.7) – There is not visible what mean grey and white bars. I suppose that gray is the GAL and white is the PAL. The correction is needed.
17. Line 276 – ‘… properties of biodiesel….’ – Authors are asked to explain if they used for calculations the biodiesel of chemical composition obtained in this study, or other.
18. Line 309 – Authors are asked to explain why they prepared the alginate solution exact at 40 °C. How long it took to obtain homogeneous solutions of blended hydrogels?
19. Line 311 – The information about dimensions of received hydrogel beads is missing.
20. Line 358 – ‘The oil … methods.’ – This sentence is unclear. Please rephrase it.
21. Line 367 – ‘enzyme units’ – instead of this should be ‘activity units’
22. Lines 371-372 – The definition of immobilisation yield is unclear rephrase the sentence). Moreover, due to typical research on enzyme immobilisation it is more preferred to determine the immobilisation yield rather from catalytic activity than from protein content.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript is poorly written and most of the corrections were not adequately addressed. The introduction wasn´t corrected and results were unclearly discussed. Furthermore, most of the improvements suggested were not take into account.

Reviewer 2 Report

Dear Authors,

Thank you very much for revision of your manuscript. Nevertheless  there are still some unresolved / unexplained issues (addressed in the first round of the review) that should be obligatory explained/improved before the manuscript could be recommended for further steps of publication process. My comments and requests for clarification /improvement are described in detail below.

 

Overall comments that should be taken by Authors for the consideration and answer:

1. Despite my first request the main aim of the study is still not clearly defined. In the introduction section (lines 91-99) reader could find only the description of what kind of experiments were carried out in presented work. I would like again ask to emphasize the purpose and significance of the research in more explicit.
2. The Conclusions section is still too general (lines 442-448). Authors did not change in this section a single word. There is no information about the results achieved by the Authors at all. It should be improved with the comparison of the results with current literature reports, that could allow to prove the significance of performed study.
3. Unfortunately, the English style and grammar has not been improved either. Therefore the extensive editing of English language and style is still required.

 

Detailed remarks to the manuscript

1. Line 49 – I repeat my remark from the first round of review

Authors write about ‘hydrophobic support’ but their study concerns hydrogel materials (alginate, pectin, gelatin) that are hydrophilic in their nature. I still don’t understand why Authors write about ‘hydrophobic support, because this is not the aim of presented study.

Please explain the reason why You write about hydrophobic carrier or remove this confusing information.

2. Line 343-344 – I repeat my remark from the first round of review

Authors are asked to explain why they prepared the alginate solution exact at 40 °C. How long it took to obtain homogeneous solutions of blended hydrogels?

I am not sufficiently satisfied with explanation given by Authors in the response for the first round of my review.

From my research experience with alginate it is really hard to prepare 2% water/buffer solution of alginate even in 1h at 60 °C. In my opinion 20 min at 40 °C is not enough to prepare homogeneous solution of alginate in given concentration.

Please give me the reasonable explanation / literature references to evidence why You used such low temperature.

Comments for author File: Comments.pdf