Design Optimization of a Tray Bioreactor for Solid-State Fermentation: Study of Process Parameters through Protein Modification of By-Products

Round 1

Reviewer 1 Report

 

In the present study, the authors investigated the design of a tray bioreactor for solid-state fermentation applying Rhyzopus oryzae to oilseed meals as substrates. The article provides a lot of relevant information and will certainly contribute to the development of this area of knowledge. However, some improvements need to be made.

·       Line 111 - Just mentioning the reference of the sensors is not enough. It must be described what type of sensor it is to facilitate reading.

·        Line 150 - What is the initial moisture content of the dry substrate? and before fermentation? This information must be added!

·        line 153 - How was the air humidity controlled? Why was it necessary to add supersaturated saline solution?

·        Line 159 - What is the objective of inactivating the fungus? And as for the spores, is this temperature enough? Information should be further explained.

·        Why were petri dishes used and not trays? Carrying out the experiments in trays would have been much more interesting.

·        The terms and abbreviations of equations 1 and 2 must be described.

·        The results in Table 1 should be further discussed.

·        "Design of pilot scale tray reactor" section is not required and the text can be added to other sections.

These changes must be made for the article to be published!

 

 

Author Response

Thank you for your feedback. We appreciate your insights and will certainly consider making the necessary improvements.

• Line 111 - Just mentioning the reference of the sensors is not enough. It must be described what type of sensor it is to facilitate reading.

Thanks for the suggestion. The DHT22 (AM2302) is a high-performance, low-cost digital temperature and relative humidity sensor. It integrates a capacitive humidity sensor and a thermistor to measure the surrounding air.

This information has been implemented in the manuscript.

• Line 150 - What is the initial moisture content of the dry substrate? and before fermentation? This information must be added!

Thank you for the suggestion. The initial humidity value is 6.7% for both by-products. As all results are expressed as dry matter in the paper, we did not consider adding the initial moisture content of the sources. Although, it could be added when the protein content of the initial sources is explained (line 300). The moisture content before the fermentation is explained in the line 134, in the pretreatments of the by-products before the inoculation.

• line 153 - How was the air humidity controlled? Why was it necessary to add supersaturated saline solution?

The air humidity is monitored every day using the sensors. The use of a saturated salt ensures the generation of a relative humidity in the bioreactor. In this case, the salt was saturated to maintain 70% at 25ºC.

• Line 159 - What is the objective of inactivating the fungus? And as for the spores, is this temperature enough? Information should be further explained.

The objective of inactivating the fungus is to stop the fermentation process and the growth of the fungus growth, as well as to halt the proteolytic reactions that are taking place.

To ensure the effective inactivation of fungal spores, combinations of treatments are often used. The choice of the most suitable method depends on the fungal species. In this context, and based on the literature, the application of the time-temperature binomial should be sufficient to inactivate spores.

• Why were petri dishes used and not trays? Carrying out the experiments in trays would have been much more interesting.

Petri dishes were used to have repetitions within each tray to better control fermentation parameters during the process. This study represents a preliminary step for the research group, aiming to further apply this process to a tray bioreactor in a whole tray.

• The terms and abbreviations of equations 1 and 2 must be described.

Thank you for the suggestion, terms and abbreviations in these equations have been added.

• The results in Table 1 should be further discussed.

Thanks for the observation. This section has been reviewed.

• "Design of pilot scale tray reactor" section is not required and the text can be added to other sections.

This section has been included as a way of draw conclusions of the results and discussions presented in the scientific paper, and to explore prospects for designing parameters for a pilot scale tray reactor

Author Response File: Author Response.pdf

Reviewer 2 Report

# Define abbreviations upon first appearance in the text. Do it in the whole article.

# The abstract should support more quantitative results.

# Why did the authors study only the temperature and relative humidity in a tray bioreactor to optimize the fermentation process?

# The study design was good and clear

# Why 1.0 mL of sterile distilled water was added to the pellet NOT saline solution?

# Please correct the scientific name of the studied microbes. Rhizopus, not Rhyzopus. check accordingly. 

# Unify the writing of heading and subheading titles 

# Most of the experimental parts were measured by a method from the author's laboratory or taken from literature? There are not enough references mentioned for these methods.

 

Minor editing of English language required

Author Response

Thank you for your feedback. We appreciate your insights and will certainly consider making the necessary improvements.

# Define abbreviations upon first appearance in the text. Do it in the whole article. 

Thanks for the observation. We have checked the whole paper and implemented the abbreviations through it. 

# The abstract should support more quantitative results. 

Thank you for the suggestion. The abstract has been reviewed, and additional and quantitative results regarding temperature and relative humidity have been incorporated. 

# Why did the authors study only the temperature and relative humidity in a tray bioreactor to optimize the fermentation process? 

Based on the literature, the two most critical factors affecting the growth of microorganisms in solid-state fermentation are the type of substrate used and the alterations in the substrates to which they are applied.  

# The study design was good and clear 

Thank you very much for your kind feedback on the study design.  

# Why was 1.0 mL of sterile distilled water added to the pellet NOT saline solution? 

We followed the instructions of microbial supplier in the microorganism propagation. In this case, ATCC.  

# Please correct the scientific name of the studied microbes. Rhizopus, not Rhyzopus. check accordingly.  

Thank you for the revision. The error of ‘Rhyzopus’ has been corrected in the abstract.  

# Unify the writing of heading and subheading titles  

Thank you for the suggestion. The writing of heading and subheading titles have been revised following the template instructions. 

# Most of the experimental parts were measured by a method from the author's laboratory or taken from literature? There are not enough references mentioned for these methods 

Thank you for the suggestion. Several methods have been mentioned in the scientific paper for methodologies. All those methods that do not have a citation are based on internal protocols of the centre.   

Author Response File: Author Response.pdf

Reviewer 3 Report

However, my main concerns are;

 1.     The abstract should be revised to add some specific quantitative data and highlight results more interestingly providing major key findings.

 2.       Authors need to explain the significance of this work in the introduction section.

 3.       Microorganisms' names should be in italic format Rhizopus oryzae.

 4.     Why a 10^6 spores/mL of inoculum used since we usually go 10^5 spores/mL for solid-state fermentation?

 5.     The figure shows solid-state fermentation and how the culture process avoids contamination by other stray bacteria.

 6.      The water content during fermentation affects protein production and what is the water content at the end of fermentation for this experiment?

 7.     An insufficient approach related to economic analysis tray bioreactor for solid-state and among the various technologies.

 

 

Author Response

Reviewer 3

However, my main concerns are:

1. The abstract should be revised to add some specific quantitative data and highlight results more interestingly providing major key findings.

Thank you for your suggestion. Abstract has been revised and quantitative data and highlight results have been added.

2. Authors need to explain the significance of this work in the introduction section.

Thank you for your comment. The significance of this work is explained in the last paragraph; however, we have checked it and a phrase was added to describe the importance of the work.

3. Microorganisms' names should be in italic format Rhizopus oryzae.

Thank you for the review. All Rhizopus oryzae were checked and changed the error in line 377.

4. Why a 10^6 spores/mL of inoculum used since we usually go 10^5 spores/mL for solid-state fermentation?

Thank you for the commentary. Inoculum was set at 10⁶ spores/mL based on various research works [1], and also previous experiments of the research group (research paper currently on revision).

1. Sukma, A.; Anwar, H.; Ikhsanudin, A. Effect of Rhizopus Oryzae Fermentation on Proximate Composition, Anti-Nutrient Contents, and Functional Properties of Banana Peel Flour. Int. Food Res. J. 2022, 29, 1205–1214, doi:10.47836/ifrj.29.5.21.
2. The figure shows solid-state fermentation and how the culture process avoids contamination by other stray bacteria.

Thank you for your commentary. Before the inoculation, the by-product was sterilised to avoid contaminations by other fungus or bacteria.

6. The water content during fermentation affects protein production and what is the water content at the end of fermentation for this experiment?

Thank you for your commentary. The moisture at the end of the fermentation was not calculated in this experiment, as the fermented product is dried to inactivate the fungus and stabilise the product. Although, from previous experimentation of the research group, the weight loses are around 30%, all water content.

7. An insufficient approach related to economic analysis tray bioreactor for solid-state and among the various technologies.

Thank you for your commentary. Economic viability analysis was not the objective of this study, as some other studies have revised the economic feasibility of solid-state fermentation [2]. This study was focused on the influence of the process variables. It is an interesting point of view; it could be added in future works.

2. Yazid, N.A.; Barrena, R.; Komilis, D.; Sánchez, A. Solid-State Fermentation as a Novel Paradigm for Organic Waste Valorization: A Review. Sustain. 2017, 9, 1–28, doi:10.3390/su9020224.

Author Response File: Author Response.pdf