Monitoring β-Fructofuranosidase Activity through Kluyveromyces marxianus in Bioreactor Using a Lab-Made Sequential Analysis System

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, this work offers a novel method for employing a sequential injection analysis (SIA) system to track the activity of β-fructofuranosidase in a bioprocess. The research's importance in relation to the fructose syrup market and the requirement for enhanced monitoring and control procedures are both highlighted in the paper, which offers a succinct and straightforward summary of the work.

Advantages:

Innovative Methodology: The monitoring of -fructofuranosidase activity using a lab-made SIA system is an inventive solution to the existing absence of appropriate sensors for this function.

Automated Sampling: The system is a potentially useful tool for real-time monitoring since it can automatically sample from the bioreactor, increasing efficiency and lowering the need for manual intervention.

Analytical Technique: The results are more reliable since 3,5-dinitrosalicylic (DNS) is a well-established approach for enzyme activity analysis.

Being transparent About Error Rates: The paper gives a realistic evaluation of the system's performance by discussing the error rates that are achieved using the automated SIA in comparison to off-line laboratory results.

Topics for Development:

Performance Variability: The system's dependability is called into doubt, particularly at low β-fructofuranosidase activity levels, given the substantial fluctuation in enzymatic activity error (ranging from 0.07% to 20.39%). A discussion of possible causes of variability and suggested correction methods would be beneficial to the study.

Comparison and Validation: Details on the procedure for validating the SIA system's accuracy and precision would be beneficial to have. Furthermore, if available, a comparison of the system's performance with other currently used techniques for tracking β-fructofuranosidase activity might be provided.

Practical consequences: Although the study report demonstrates the SIA system's potential as a real-time monitoring tool, it would benefit from a discussion of the uses and practical consequences of this technology in the workplace and in academic contexts.

 

All things considered, this work fills a significant gap in the literature and presents a viable approach of tracking β-fructofuranosidase activity. Additional improvements and verification of the SIA system should result in significant contributions to this field of study.

Author Response

Reviewer 1

 

Comments and Suggestions for Authors

Overall, this work offers a novel method for employing a sequential injection analysis (SIA) system to track the activity of β-fructofuranosidase in a bioprocess. The research's importance in relation to the fructose syrup market and the requirement for enhanced monitoring and control procedures are both highlighted in the paper, which offers a succinct and straightforward summary of the work.

 

Advantages:

• Innovative Methodology: The monitoring of B-fructofuranosidase activity using a lab-made SIA system is an inventive solution to the existing absence of appropriate sensors for this function.
• Automated Sampling: The system is a potentially useful tool for real-time monitoring since it can automatically sample from the bioreactor, increasing efficiency and lowering the need for manual intervention.
• Analytical Technique: The results are more reliable since 3,5-dinitrosalicylic (DNS) is a well-established approach for enzyme activity analysis.
• Being transparent About Error Rates: The paper gives a realistic evaluation of the system's performance by discussing the error rates that are achieved using the automated SIA in comparison to off-line laboratory results.

 

We are grateful to Reviewer 1, for the kind comments and suggestions provided to enhance the manuscript. In fact, the listed advantages detected by the reviewer motivated us to improve the paper. Regarding the topics of development, we addressed the suggestions highlighting the text modifications in the document in blue.

 

Topics for Development:

1.- Performance Variability: The system's dependability is called into doubt, particularly at low β-fructofuranosidase activity levels, given the substantial fluctuation in enzymatic activity error (ranging from 0.07% to 20.39%). A discussion of possible causes of variability and suggested correction methods would be beneficial to the study.

 

Answer: We really appreciate this suggestion, it was included in the discussion section the possible causes of the variability in the measurements, and we argued or suggested diverse strategies to compensate or correct the error.

 

2.- Comparison and Validation: Details on the procedure for validating the SIA system's accuracy and precision would be beneficial to have. Furthermore, if available, a comparison of the system's performance with other currently used techniques for tracking β-fructofuranosidase activity might be provided.

 

Answer: We really appreciate this suggestion, we also included in the discussion section a brief discussion about the accuracy and the precision of the technique mounted on the SIA system.

 

3.- Practical consequences: Although the study report demonstrates the SIA system's potential as a real-time monitoring tool, it would benefit from a discussion of the uses and practical consequences of this technology in the workplace and in academic contexts.

 

Answer: Great! We have included a brief discussion about how Lab-Made SIA systems can be seen from academic and research point of views.

 

4.- All things considered, this work fills a significant gap in the literature and presents a viable approach of tracking β-fructofuranosidase activity. Additional improvements and verification of the SIA system should result in significant contributions to this field of study.

 

Answer: Absolutely, we agree with the reviewer, that the SIA system has many areas of opportunity regarding improvement. During the following months/years, we will be dedicated to setting up new techniques, and measuring metabolites at-line with the aim of setting-up control strategies.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

In the present manuscript, the authors developed a computer algorithm to adapt a previously developed in-line SIA system for continuous β-fructofuranosides monitoring. The manuscript is overall well-written. I only have a few minor comments:

1. Missing transition between paragraphs 3 and 4 in the introduction.

2. Missing logic flow in the introduction. Please start directly with paragraph 4 (why β-fructofuranosides are important) and follow with the previous monitoring techniques for these specific enzymes and their technical issues.

3. Please emphasize the novelty and importance of this study in the last paragraph of the introduction. In other words, why the proposed system can address the issues of the previously used techniques? It is not clear if an SIA system has never been developed to monitor β-fructofuranosides or if the developed SIA system has never been integrated into a computer algorithm. (I see that the authors discussed the literature review and the novelty of the proposed system in the discussion. That needs to be moved to the introduction.)

4. The methods section is overall well-stated. However, it is not clear the volume needed for each sample and reagent usage (for both sample analysis and system cleaning). And how are they compared to the offline laboratory measurements?

5. Some of the materials in the discussion section seemed inappropriate to me. The first two paragraphs belong to the methods section, and the first half of paragraph 3 belongs to the introduction.

Comments on the Quality of English Language

The manuscript is overall well-written.

Author Response

Reviewer 2

Comments and Suggestions for Authors

In the present manuscript, the authors developed a computer algorithm to adapt a previously developed in-line SIA system for continuous β-fructofuranosides monitoring. The manuscript is overall well-written. I only have a few minor comments:

 

We are grateful to Reviewer 2, for the kind suggestions to enhance the manuscript. We have addressed and corrected all the suggestions highlighting text modifications in blue. The introduction was rewritten starting with the importance of the yeast K. marxianus, the relevance of β-fructofuranosides, the strategies to detect enzyme activity, the problem statement, and the proposed way to solve it.

 

Reply to specific Reviewer concerns or suggestions.

 

1. Missing transition between paragraphs 3 and 4 in the introduction.

 

Answer: This issue was addressed along with the second reviewer's suggestion, taking care to provide appropriate transitions among all the paragraphs in the introduction section.

 

2. Missing logic flow in the introduction. Please start directly with paragraph 4 (why β-fructofuranosides are important) and follow with the previous monitoring techniques for these specific enzymes and their technical issues.

 

Answer: We really appreciate this suggestion, we have started the introduction section describing the yeast Kluyveromyces marxianus, followed by the relevance of β-fructofuranosides; as well as the diverse monitoring procedures to detect these enzymes.

 

3. Please emphasize the novelty and importance of this study in the last paragraph of the introduction. In other words, why the proposed system can address the issues of the previously used techniques? It is not clear if an SIA system has never been developed to monitor β-fructofuranosides or if the developed SIA system has never been integrated into a computer algorithm. (I see that the authors discussed the literature review and the novelty of the proposed system in the discussion. That needs to be moved to the introduction.)

 

Answer: We thank the reviewer for this relevant suggestion. Now the closing section in the introduction emphasizes the novelty and importance of the study.

• SIA systems have been used by other authors to detect β-fructofuranoside activity; however, the samples were determined indirectly from offline samples. Our technique set up in the SIA system detects enzyme activity from samples taken directly from the bioreactor using a proper sampling probe with a filter to then be analyzed immediately by the SIA.
• We developed and reported a lab-made system (Pliego,2015), that was used to detect lipase and esterase activity. In this document, we describe some generalities of that lab-made SIA system such as software components, the graphical user interface, and the modifications performed to determine β-fructofuranoside activity.

 

4. The methods section is overall well-stated. However, it is not clear the volume needed for each sample and reagent usage (for both sample analysis and system cleaning). And how are they compared to the offline laboratory measurements?

 

Answer: One second of operation of injection/suction in the SIA system for this methodology was set to a flow rate of 20 mL/s, that information was shown in section 3.8.1 Calibration curve. However, we have moved this information to the Methodology section. From the operation time in each sequence (last column of Tables 1 and 2) and the flow rate, it is possible to calculate the volume used in each sequence.

The comparison between the offline and at-line measurements is shown in the discussion section when the accuracy of the method is described.

 

5. Some of the materials in the discussion section seemed inappropriate to me. The first two paragraphs belong to the methods section, and the first half of paragraph 3 belongs to the introduction.

 

Answer: we are sorry about this inconvenience; we have moved most of the information in those paragraphs to the introduction section.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors designed a LabVIEW™ algorithm to control a class of lab-made SIA system. The device was able to determine the enzymatic activity of β-fructofuranosidase, showing a standard deviation below 7% at activities lower than 11 U/ml with standard deviations below 4% and errors below 21% in comparison with the measurement performed offline. As a whole, the work can be published after minor revisions as follows:

1 Fig 1, F part is missing, please add.

2 Fig2, the number order should follow the flow chart from the start to the end.

3 L12 in the 1st page, 20.39% should be revised to 20%.

4 There are a number of grammar errors, please check and correct throughout. For example, ‘The setting panel shown in Figure 5B it is intended to load and save the diverse 268 methodologies created in the SIA system’ has error in grammar. Please delete ‘it’.

5 Please analyze the possibility of transfering the proposed method into an advanced and non real time technique.

Comments on the Quality of English Language

Mostly acceptable, with some errors in grammar.

Author Response

Reviewer 3

Please review the attached Word document, in which we have included some figures.

We are grateful to Reviewer 3, for the kind suggestions provided to enhance the quality of the manuscript. We remarked the corrections in the manuscript using blue color.

Comments and Suggestions for Authors

The authors designed a LabVIEW™ algorithm to control a class of lab-made SIA system. The device was able to determine the enzymatic activity of β-fructofuranosidase, showing a standard deviation below 7% at activities lower than 11 U/ml with standard deviations below 4% and errors below 21% in comparison with the measurement performed offline. As a whole, the work can be published after minor revisions as follows:

1 Fig 1, F part is missing, please add.

 

Answer: We really appreciate the reviewer's comment. Please notice that the component “F” appears in the original Figure 1, as can be observed in the computer image. However, from the reviewer's comment, we realized that the order of the components may be confusing, so we modify the sequence of elements and enlarged the letters, starting with the letter A, and so on, as can be seen in the new modified Figure 1.

           Consequently, the legend of Figure 1 was modified as:

Figure 1. Main components of the SIA System with UV/visible spectrophotometer; A) graphical user interface, B) peristaltic pump, C) holding coil, D) multi-position valves, E) reaction coil, and F) flow cell.

 

2 Fig2, the number order should follow the flow chart from the start to the end.

 Answer: The reviewer is right, the order of the components in Figure 2 is confusing. To solve this issue, we replaced numbers with letters, and then we ordered the elements following the flow chart from left to right.

       

    

 

Label

Figure 2. Specific components of the Lab-Made used to determine β-fructofuranosidase activity. A) carrier, B) retentions chamber, C) heating chamber, D) and E) mixer chambers, F) bioreactor and sampling probe, G) cooling chamber, H) substrate, I) reagents, J) incubation chamber, K) spectrophotometric detector, and L) waste container.

 

3 L12 in the 1st page, 20.39% should be revised to 20%.

Answer: We have corrected the text in the discussion section to state that “the maximum error in this investigation was 20.39\%, instead of about 20%”.

4 There are a number of grammar errors, please check and correct throughout. For example, ‘The setting panel shown in Figure 5B it is intended to load and save the diverse 268 methodologies created in the SIA system’ has error in grammar. Please delete ‘it’.

Answer: We apologize for the grammar errors in the document and the Figure. We carefully reviewed the document to detect and correct those errors. We also corrected the grammar errors in the different subsections of Figure 5.

5 Please analyze the possibility of transfering the proposed method into an advanced and non real time technique.

Answer: One advantage of our lab-made SIA system is the capability to be used as an at-line real-time monitoring system taking samples out in an automatic way from the bioreactor using a specialized sample probe with a ceramic filter. However, the SIA system could be operated in nonreal-time environments, for example, the SIA system could be located far from the bioreactor or even in another laboratory.