In Situ Entrapment of Catalase within Macroporous Cryogel Matrix for Ethanol Oxidation: Flow-through Mode versus Batch Reactor

Round 1

Reviewer 1 Report

This paper is interesnting. It deals about etanol oxidation using  with catalase entrapped in a catlyst. It is a technical report, the authors must indicate the scientific knowledge generated from the obtained resilts. There are issues with English. The paper can be published should the authors attend the following observations.

1.- Eigth authors is a big number of authors, if this is allowed by the review then this is OK.

2.- The abstract has repetitions.

3.- page 1.- “supports has been demonstrated the high activity”….please correct….”supports have been demonstrated the high activity”

4.- Page 2.- “According to the classification of biocatalytic processes immobilized-enzyme 47 catalysis has been attracting attention”…..please correct, place a comma…..”According to the classification of biocatalytic processes, immobilized-enzyme 47 catalysis has been attracting attention”

5.- Page 2.- “solvent play the role of porogens after defrosting”…..please revise if the Word “progens” is correct in this context.

6.- page 3.-  It seems this paper is the report of only exerimental work.

7.- page 3.- “Such large pores provide for the free flow of liquid substrate and oxidation agent under gravity and at hydrostatic pressure.”…..please rework this text. Revise the Word, “provide”….probably ….”Such large pores are  adequte for the free flow of liquid substrate and oxidation agent under gravity and at hydrostatic pressure.”

8.- Page 4.- “and 221 U×mL-1”….please give the meaning of “U”.

9.- Page 5.- “It is noticeable that the maximum activity of gellan gel-immobilized catalase [39] 157 have demonstrated at a pH 6.5.”……please corect….”It is noticeable that the maximum activity of gellan gel-immobilized catalase [39] 157 was demonstrated at a pH 6.5.”

10.- page 5.- “ the yield of acetaldehyde reached up to 89.5% (Figure 5).”……please clarify if it is yield or conversión, in figure 5 the ordinates are conversión.

11.- page 5.- “the cryogel is preferentially in the swollen state and the pores are more accessible for”….please rework this text, mainly if the Word “swollen” is correct in this context

12.- page 5.- “The activity of cryogel-encapsulated catalase is higher at low temperature (Figure  6). Probably”…..please note that in figure 6 the yield is reported and not activiyt. Activity and yield are different concepts.

13.- page 5.- “Increasing of temperature can cause shrinking of cryogel matrix retarding the effective diffusion coefficient of substrate to surface area due to lower internal mass transfer resistance.”….please correct….”Increasing of temperature can cause shrinking of cryogel matrix diminishing  the effective diffusion coefficient of substrate to surface área, due to lower internal mass transfer.”, ….please note that the “lower internal mass transfer resistance” is to enhance the mass transport. If the resistance increase, the mass transport is reduced.

14.- Page 6.- Please explain the meaning of “antibatically”

15.- Page 6.- “The oxidation of ethanol can conditionally be divided into 3 stages. The first stage is up to 4 min where the process is slow (induction period). The second stage is lasted between 4-10 min where…”….please correct….”The oxidation of ethanol can be divided into 3 stages. The first stage is up to 4 min where the process is slow (induction period). The second stage lasts between 4-10 min where”

16.- Page 6.- legend of figure 7…..”Figure 7. Dependence ethanol conversion (1) and yield”…..please correct….”Figure 7. Dependence of ethanol conversion (1) and yield”

17.- Page 8.- “The value of the coefficient of determination (R2 219 = 0.9811) indicates….”….please correct…..”The value of the coefficient of correlation (R2 219 = 0.9811) indicates”

18.- Page 8.-“The activation energy was equal to Еa = 7.31 kJ×mol-1 234 that is two times less than the value for the activation energy calculated for the decomposition of H2O2 (Ea = 14 kJ×mol-1 235 )”….please  correct…”The activation energy was equal to Еa = 7.31 kJ×mol-1 234 that is one half of the value of the activation energy calculated for the decomposition of H2O2 (Ea = 14 kJ×mol-1 235 )”

19.- page  9.- If tables and figures give the same information, The tables are  necessary?. Please see Figure 11 and table 3.

20.- page 9.- “was studied in batch type reactor at 20 °C with hydrogen peroxide,”….please provide the dimensions of the reactor and the necessay details.

21.- Figure 10, why the slope is positive?, Then te activation energy is negative?, what is the meaning of the negative activation energy?

22.- page  10.- “The yield of acetaldehyde, according to GC analysis, was high for  both powdered cryogel (96.7%) and monolithic cryogel (97.7%). However, the yield and  conversion of ethanol decreased by 1.6 times when the experiments were performed in air.”….please give the reasons why these facts happen.

23.- page 10.- “the same cryogel sample in 5 times successive oxidation reactions”….please correct….” the same cryogel sample in 5 successive oxidation reactions”

24.- page 10.- In table 4, why the contact time is not the same for each run? The time is less in sucesive runs. The diminution in conversión is 12.9% and it is not possible neglect it, but it must be considered.

25.- page 10.- In table, what is the meaning of each columna? For example the second, third and dourth columns correspond to H2O2, but what is the diference among them?

26.- page 10,. “for oxidation of ethanol given in Table 7.”….please correct….” for oxidation of ethanol are given in Table 7.”

27.- page 11.- Figure 12 is not shown….”The reaction mechanism proceeds in two steps (Figure 12).”.Reaction I  is not shown,

29.- page 12.- “catalase, 4.17 is conversion coefficient of catalase to arbitrary unit”…..please correct….”catalase, 4.17 is conversion coefficient of catalase with arbitrary unit”

30.- Page 13.- “Oxidation of ethanol was carried out on a flow-through catalytic reactor”…..please crrect….”Oxidation of ethanol was carried out in a flow-through catalytic reactor”

31.- page 13.- Please confirm that the reactors tubular and batch are comparable in order to judge the results.

32.- Where can I see the comparison between batch and tubular?, in which figure?.

33.- Table 7 looks that those reported in the text books,and if it is not associated with the results this table 7 is uselessin in this paper

33.- Conclusions are huge, extremly long, and they are not conclusions. Only description of the resuts, the text is like a summary of the paper. This material is a technical report, please indicate what is the scientific generated knowledge.

34.- Suplementary material con not be downloaded from the povided link

 

Te majority of my observations is about English. Englishh should be improved

Author Response

The authors express their gratitude to the reviewers for their careful review of the manuscript and the comments and suggestions made to the manuscript to improve its quality and scientific soundness. All changes in revised manuscript are highlighted in red.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript “In situ Entrapment of Catalase within Macroporous Cryogel Matrix for Ethanol Oxidation: Flow-through Mode versus Batch Reactor” contains interesting observations and ideas but overall needs significant improvement. The catalytic oxidation of ethanol to acetaldehyde is of interest in particular if air is used. The process should be compared to the Wacker Oxidation of ethylene to acetaldehyde.

Several recent review go into detail on how to describe this type of flow chemistry, please see: L. Tamborini, P. Fernandes, F. Paradisi, F. Molinari, Flow bioreactors as complementary tools for biocatalytic process intensification, Trends Biotech. 36 (1) (2018) 73–88, https://doi.org/10.1016/j.tibtech.2017.09.005. J. Britton, S. Majumdar, G.A. Weiss, Continuous flow biocatalysis, Chem. Soc. Rev. 47 (15) (2018) 5891–5918, https://doi.org/10.1039/C7CS00906B. Coloma, J.; Guiavarc’h, Y.; Hagedoorn, P.-L.; Hanefeld, U. Immobilisation and flow chemistry: Tools for implementing biocatalysis. Chem. Commun. 2021, 57, 11416–11428. https://doi.org/10.1039/D1CC04315C  Sokaˇc Cvetni´c, T.; Šali´c, A.; Benkovi´c, M.; Jurina, T.; Valinger, D.; Gajdoš Kljusuri´c, J.; Zeli´c, B.; Jurinjak Tušek, A. A Systematic Review of Enzymatic Kinetics in Microreactors. Catalysts 2023, 13, 708. https://doi.org/10.3390/catal13040708 or the review Chem. Eur. J. 2022, 28, e202103607 doi.org/10.1002/chem.202103607 These reviews might help to describe the interesting science of the manuscript better.

There are several points that need clarification. What actually is the catalyst? If H2O2 is cleaved to O2 by catalase and this then oxidizes the ethanol that would make sense and it would match the batch reactions. The authors should clarify this.

But there are also other questions: How is the reaction followed? The chromatogram in fig.3 is unclear. What Voltage is measured? Is there an internal standard?

When the experimental procedure is evaluated then Line 331 it should be added what the concentration of the phosphate buffer is and what the counter ion is. In 3.3 a titration is described but please state clearly what is titrated.

In 3.5.1 alcohol and H2O2 are used 1:1 by volume. But ethanol is pure and H2O2 is a 0.07 molar solution in water. What does the conversion then refer to in all the tables?

In 3.5.2. hexane is added to the mixture. If hexane, oxygen and a catalyst are mixed this is a potentially explosive mixture. Could the authors explain why hexane is added?

In the text under fig. 4 does 10 mg catalase stand for 10 mg immobilized catalase or 10 mg catalase plus the carrier.

Page 6 fig 6 could the decrease in yield be due to the high volatility of acetaldehyde. How are the mass balances?

In fig. 8 what explains the fluctuations in conversion over time?

Table 4 please add the reaction time.

The reaction schemes should actually not be in the SI as they are essential to understand the catalysis taking place.

 

needs some improvement

Author Response

The authors express their gratitude to the reviewers for their careful review of the manuscript and the comments and suggestions made to the manuscript to improve its quality and scientific soundness. All changes in revised manuscript are highlighted in red.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The revised manuscript is now fine and can be accepted