A Theoretical and Experimental Study for Enzymatic Biodiesel Production from Babassu Oil (Orbignya sp.) Using Eversa Lipase

Round 1

Reviewer 1 Report

This is a theoretical and experimental work for biodiesel from Babassu oil, which is very important in biomaterial energy . The corresponding comments are as follows:

1.     FFA (free fatty acid) should be explained at the first time.

2.     In Table 1 the commas should be dots for the conversion and S/R? Similar errors in the text should also be corrected

3.     In Table 1, S/R might be S/N according to the text. Similar errors for equation (3) in Page 14.

4.     Orthogonal experimental method was used in the paper to explore the optimization of reaction condition. In general, orthogonal experiment is only to be used after the single factor analysis experiment, which get the effects of different factors on the conversion and S/N. Or, the orthogonal experiment might not get the optimal results.

5.     After orthogonal experiment, statistical analysis is needed to evaluate every factor and for the combination of the possible optimal factor to get a further improvement in performance.

In summary, if the authors could revise as the suggestion above, I recommend it to be published.

Author Response

Dear,

We would like to thank the reviewers for their crucial and constructive criticism, which significantly improved our work's scientific quality. Please find below the answers to their comments and questions. For ease of reference, all modifications were marked in the revised manuscript version.

This is a theoretical and experimental work for biodiesel from Babassu oil, which is very important in biomaterial energy. The corresponding comments are as follows:

Thank you for your comments and contributions.

1. FFA (free fatty acid) should be explained at the first time.

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript. The explanation of free fatty acids (FFA) was incorporated in the abstract (lines 27-28).

2. In Table 1 the commas should be dots for the conversion and S/R? Similar errors in the text should also be corrected

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript.

3. In Table 1, S/R might be S/N according to the text. Similar errors for equation (3) in Page 14.

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript.

4. Orthogonal experimental method was used in the paper to explore the optimization of reaction condition. In general, orthogonal experiment is only to be used after the single factor analysis experiment, which get the effects of different factors on the conversion and S/N. Or, the orthogonal experiment might not get the optimal results.

Thank you for your comments and contributions. The main focus of this work is the theoretical study of the enzymatic synthesis of babassu methyl esters. Thus, the parameters evaluated were selected according to data obtained in the literature:

https://doi.org/10.4028/www.scientific.net/AMR.791-793.196

https://doi.org/10.1016/j.apenergy.2011.04.041

https://doi.org/10.1080/15567036.2018.1495778

5. After orthogonal experiment, statistical analysis is needed to evaluate every factor and for the combination of the possible optimal factor to get a further improvement in performance.

Thank you for your comments and contributions. During the work, the individual influence of the parameters on the process was evaluated in item 2.2.

 

Reviewer 2 Report

The authors report a combined theoretical and experimental study to investigate biodiesel production through hydroesterification. The authors use Taguchi's experimental design to obtain the base dataset and utilize that for process optimization. The "optimized" conditions yielded a high conversion in accordance with the predictions. Overall I found the work interesting and the approach sound. I would therefore recommend the work for publication, once the following question in addressed.

What are the typically error bars for the measurements and how replicable are the experiments? The experiment at the optimized conditions yielded a conversion of 95.76% vs. 98.64% as predicted by the model. Is this within the error bars of the experiments?

Author Response

Dear ,

We would like to thank the reviewers for their crucial and constructive criticism, which significantly improved our work's scientific quality. Please find below the answers to their comments and questions. For ease of reference, all modifications were marked in the revised manuscript version.

1. The authors report a combined theoretical and experimental study to investigate biodiesel production through hydroesterification. The authors use Taguchi's experimental design to obtain the base dataset and utilize that for process optimization. The "optimized" conditions yielded a high conversion in accordance with the predictions. Overall I found the work interesting and the approach sound. I would therefore recommend the work for publication, once the following question in addressed.

Thank you for your comments and contributions.

2. What are the typically error bars for the measurements and how replicable are the experiments? The experiment at the optimized conditions yielded a conversion of 95.76% vs. 98.64% as predicted by the model. Is this within the error bars of the experiments?

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. The experiments exposed in this work were conducted with a margin of error of 1%. Therefore, under optimal conditions, an error of 1% is expected. It is worth noting, as mentioned between lines 202 to 208, biocatalysts can quickly lose their catalytic activity, as they are more sensitive to external factors than chemical catalysts, so this small discrepancy between the actual and predicted conversion is expected. Finally, it is important to note that the theoretical conversion was validated by means of gas chromatography. All exposed experiments are reproducible.

Reviewer 3 Report

The research was aimed to evaluate  biodiesel production from Babassu oil (Orbignya sp) using Eversa lipase. The statistical method was adopted to evaluate the effectiveness of the enzyme in the production. In addition, computational method was applied to study the interaction between enzyme and the lipid as coordination reaction approach in the mechanism. The results is quite interesting, but there are some issues need to be resolved before publication:

 

1. The future perspective of this research should be mentioned in the introduction part, especially the main reason for optimizing the enzyme and its computational modelling. What is the consideration to do this??

 

2. The numbers are written by using comma (,) not point (.), please edit throughtout the manuscriot.

 

3. There are two parameters: Signal to noise (S/N) ratio and S/R (in Table 1). What is S/R??

 

4.  What is the urgency of pretein homology in this study? In addition, in line 243 authors mentioned that The ligands showed good energetic affinity, and oleic acid showed a higher binding 

affinity with the catalytic site. What is the classification for good or  poor classification based on binding energy? 

 

5. The reaction was conducted by using lipid: methanol ratio of between 1:1 to 1: 9. The ratio of methanol is higher than lipid. How about the interaction of enzyme with methanol? Why it is not evaluated?? 

Author Response

Dear,

We would like to thank the reviewers for their crucial and constructive criticism, which significantly improved our work's scientific quality. Please find below the answers to their comments and questions. For ease of reference, all modifications were marked in the revised manuscript version.

Comments and Suggestions for Authors

The research was aimed to evaluate biodiesel production from Babassu oil (Orbignya sp) using Eversa lipase. The statistical method was adopted to evaluate the effectiveness of the enzyme in the production. In addition, computational method was applied to study the interaction between enzyme and the lipid as coordination reaction approach in the mechanism. The results is quite interesting, but there are some issues need to be resolved before publication:

Thank you for your comments and contributions.  

1. The future perspective of this research should be mentioned in the introduction part, especially the main reason for optimizing the enzyme and its computational modelling. What is the consideration to do this?

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript. Perspectives were included between lines 83 to 87.

2. The numbers are written by using comma (,) not point (.), please edit throughtout the manuscriot.

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript.

3. There are two parameters: Signal to noise (S/N) ratio and S/R (in Table 1). What is S/R??

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript.

4. What is the urgency of pretein homology in this study? In addition, in line 243 authors mentioned that The ligands showed good energetic affinity, and oleic acid showed a higher binding affinity with the catalytic site. What is the classification for good or  poor classification based on binding energy? 

Thank you for your comments and contributions. All suggestions were incorporated into the new version of the manuscript. Changes are in the revised manuscript. The crystal structure for lipase Eversa was not available in the PDB database, because of this, the lipase homology model was developed. Corrections made are marked in yellow.

5. The reaction was conducted by using lipid: methanol ratio of between 1:1 to 1: 9. The ratio of methanol is higher than lipid. How about the interaction of enzyme with methanol? Why it is not evaluated?? 

Thank you for your comments and contributions. The interactions between the alcohol and the catalytic site were not evaluated, since the results obtained in the synthesis step suggest that the molar ratio presents low interference in the conversion. Furthermore, the objective of the theoretical study is to provide a structural view of the interactions between Eversa and fatty acids.