Continuous Production of DHA and EPA Ethyl Esters via Lipase-Catalyzed Transesterification in an Ultrasonic Packed-Bed Bioreactor

Round 1

Reviewer 1 Report

This manuscript demonstrates the solvent-free transesterification of DHA and EPA using an immobilized lipase in a packed-bed bioreactor. The authors coupled ultrasonication with their reactor and found that under batch reaction, ultrasonication enables more efficient production of DHA and EPA ethyl esters from DHA + EPA concentrate and ethyl acetate. The study further demonstrated a continuous operation of the reactor for 5 days and the results indicated a robust and highly effective production of DHA and EPA ethyl esters, suggesting the industrial applicability of the enzyme used. While the results of batch experiments were also supported by mass transfer/kinetic models, the study needs major improvements to warrant its publication in the journal. A few comments for the authors are appended below:

1. The results of the continuous operation of packed-bed reactor indicated no substantial improvement in the recovery of DHA and EPA ethyl esters when using ultrasonication. In other words, continuous operation rules out the effectiveness of ultrasonication observed in batch experiments and this could eventually limit the industrial applicability of ultrasonication.
2. While the title of the manuscript seemingly emphasizes the effectiveness of ultrasonication in the production of DHA/EPA ethyl esters, the authors have only tested the operation of ultrasonication under 1 condition (37 kHz, with 100% output power). Comparison of different operation conditions will be needed if the authors would like to emphasize this part of the results. Otherwise, it would be better if the results obtained different operation mode (continuous vs. batch) are more emphasized/discussed upright throughout the manuscript. i.e., Why was there no substantial difference between ultrasonication-amended vs. -not amended treatments under continuous operation? This question must be addressed somewhere in the manuscript.
3. Even though the batch results suggested an increase in the conversion efficiency when using the ultrasonication, the authors need to provide more context to the significance of this. For example, in Table 1, treatment No. 10 indicates that the % conversion increased from 86.6% to 93.0% when using the ultrasonication with flow rate of 5 ml/min and the substrate (DHA+EPA) concentration of 500 mM. Is this increment meaningful enough? Roughly 6% increase in the % conversion may not be significant when considering energy inputs associated with the newly added ultrasonication process, especially in an industrial-scale operation.
4. (Lines 90-91) This sentence has been repeated twice.

Author Response

See attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

The continuous production of DHA and EPA ethyl esters, in a packed-bed reactor, by using an immobilized lipase presented in this manuscript can be of interest to researchers in this area. The overall results show the benefits of ultrasonication in increasing the reaction rate. However, my main concern is about the kinetic part, it is unclear, and it needs improvements:

(1) The title is unnecessarily long. It should be re-written

(2) Was it used as substrates a mixture of DHA, EPA, and ethyl acetate? If so, why only two substrates (A and B) are considered in the mathematical model? The kinetic parameters should be determined for each substrate. It is acceptable to not consider EA in the equation since it is saturated, but the kinetic constants for DHA and DHA must be determined separately.  

(3) Today should not be acceptable to use a linearization approach for solving the non-linear equation of Michaelis-Menten (MM). There is much software available for solving directly non-linear equations. Of course, the Lineweaver-Burk approach still has a pedagogical relevance but for research, there are advanced tools.

Author Response

See attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed the comments raised by the reviewers and the manuscript now looks acceptable for publication. Please make sure to double check the proof for any minor grammar errors (for example, Line 318). 

Author Response

The authors have addressed the comments raised by the reviewers and the manuscript now looks acceptable for publication. Please make sure to double check the proof for any minor grammar errors (for example, Line 318). 

A: We have corrected on L. 318. Our manuscripts have also been checked by a native English-speaking colleague.

 

Thank you again. We greatly appreciate your precious time for improving our manuscript.

Reviewer 2 Report

I do not recommend the publication of this manuscript in its current form. The authors could not overcome the main flaws regarding the kinetic characterization. I understand the economic limitations expressed by the authors, but the simplification of the mathematical model is not convincing. Furthermore, the use of only 4 to 5 points to determine the kinetic constants of this system does not provide enough experimental evidence. In summary, the kinetic study presented in the manuscript is unacceptable. If the authors have financial and technical constraints to perform an adequate kinetic study, they can skip that section, and instead do a more in-depth analysis of yield coefficients and productivity. 

Author Response

I do not recommend the publication of this manuscript in its current form. The authors could not overcome the main flaws regarding the kinetic characterization. I understand the economic limitations expressed by the authors, but the simplification of the mathematical model is not convincing. Furthermore, the use of only 4 to 5 points to determine the kinetic constants of this system does not provide enough experimental evidence. In summary, the kinetic study presented in the manuscript is unacceptable. If the authors have financial and technical constraints to perform an adequate kinetic study, they can skip that section, and instead do a more in-depth analysis of yield coefficients and productivity. 

A: Thanks for your comments and suggestions that allowed us to greatly improve the quality of the manuscript. As you proposed, we kept all items in the Eq. (5) and developed an apparent Michaelis-Menten equation as Eq. (7) to solve the kinetic study (L.299-304). Since solvent-free system is a special case, the apparent Michaelis-Menten equation can be applied. We also provided several examples (ref. 45-49) using Lineweaver–Burk plot to solve the kinetic parameters in the solvent-free system and in these citations only 4-5 experimental points were used (L.305-312). The Lineweaver–Burk plot (Fig.6) showed there was no substrate inhibition within the range of our experiments, so the apparent Michaelis-Menten equation can be applied to our range of experimental substrate concentration. We also removed the extent axes of the regression in Figure 6 to avoid the extended part might have substrate inhibition. In the packed-bed bioreactor, the substrate was reacted in the presence of excess enzymes. In order to further examine the effect of ultrasonication, we performed enzyme kinetic studies in the batch reaction with trace amount of enzyme. Therefore, we moved section 2.1 (old) to section 2.5 (new) and change its title to “Evaluate the ultrasonication effect by the kinetic model in the batch reaction”. In addition, we have also successfully developed an apparent Michaelis-Menten equation that can be used to compare the efficiency of lipase-catalyzed reactions under different reactor operations.

Thank you again. We greatly appreciate your precious time for improving our manuscript.

 

Added Reference

45      Aljawish, A.; Heuson, E.; Bigan, M.; Froidevaux, R. Lipase catalyzed esterification of formic acid in solvent and solvent-free systems. Biocatalysis and Agricultural Biotechnology 2019, 20, 101221.

46. Jaiswal, K.S.; Rathod, V.K. Green synthesis of amyl levulinate using lipase in the solvent free system: Optimization, mechanism and thermodynamics studies. Catalysis Today 2021, 375, 120-131.
47. Xu, C.; Zhang, H.; Shi, J.; Zheng, M.; Xiang, X.; Huang, F.; Xiao, J. Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system. Ultrasonics Sonochemistry 2018, 41, 120-126.
48. Bayramoğlu, G.; Hazer, B.; Altıntaş, B.; Arıca, M.Y. Covalent immobilization of lipase onto amine functionalized polypropylene membrane and its application in green apple flavor (ethyl valerate) synthesis. Process biochemistry 2011, 46, 372-378.
49. Mahapatra, P.; Kumari, A.; Kumar, G.V.; Banerjee, R.; Nag, A. Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica. Biocatalysis and Biotransformation 2009, 27, 124-130.