Harnessing Packed-Bed Bioreactors’ Potential in Solid-State Fermentation: The Case of Beauveria bassiana Conidia Production
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study aimed to produce conidia using Beauveria bassiana packed-bed bioreactor and tray bioreactor. Though the study presents reasonable findings, the following queries should be addressed.
Highlights: include the optimization conditions.
Line 56: SSF does not need either water or produce wastewater - Correct it to SSF needs less water.
Line 60: In the case of biopesticides, however, this problem may have no sense if the target compound with biopesticide properties is effective in soil, since it can be applied as compost, with no purification needed [10] - This contradicts the earlier sentence. Here, the authors are stating only the challenges. Please delete it.
Line 65: This sentence is complex. Break the sentence into a simple form.
Nothing is mentioned in the methodology about the details of the type of DoE used. However, the results present the DoE results. Include the type of DoE used, the rationale behind selecting the factors and present the DoE with the combination of runs in the form of a table.
Table 2. DoE 1 and 2 performed batches. DoE 1 and DoE 2 parameters are separated by an horizontal bar. - I couldn’t see any horizontal line separating the two DoEs and the table has only one DoE.
Line 271: This contaminant was not found in previous fermentations using the same substrate - This indicates the experiment was not conducted properly.
Line 278: beer draff was selected alongside rice husk for further experiments - Justify how the rice husk addition is going to control the contamination as Aspergillus is known to grow on rice husk.
The authors frequently used that the parameters were maintained as found in the previous optimization experiments - whether they were from their experiments or from the literature. Clarify it and include the references.
Figure 4: Legends are missing.
Line 354: In the present study, we present an optimized fermentation process for production of fungal conidia for Beauveria bassiana using the packed-bed bioreactor configuration - Details of the optimization are missing.
The importance of producing conidia is missing in the manuscript. Include it either in the introduction or in the discussion.
Author Response
See attached file.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsLine 39, consider removing “circular” before CBE.
Line 102 is missing the space between value and unit “0.5L”
ºC is not the correct symbol for degrees Celsius, review the entire document where the symbol is used.
Relative to table 1, is it not understood how the mixtures with RH were defined, so that they maintain a similar value between each substrate? So that there was an adequate gas transfer rate?
Despite being a screening, the fungus did not grow with the soy residue, and they comment that it was contaminated by batteries due to the high rate of O2 consumption, but the test should have been repeated, since substrates or parameters cannot be ruled out. a study for laboratory errors or contaminations.
They propose using 2 optimization designs, which do not mention which ones are used to understand dependent and independent variables. It is also not clear why 2 independent designs with different variables are used and not a single design with all the variables to have a correct integration of all the variables.
What conditions were set in design 1 of Temperature, C/N and MC, and in design 2 what were the MC, IC and AF conditions?
The analysis of the optimal point of each design is not presented, nor is any data or graph, nor is it presented what was the optimal point of each design, or the combined one.
Line 262, do they mention the optimal conditions obtained in reactor 6 and reactor 9? These are not the optimal conditions; they are the maximums found in the design. Whoever guarantees that the mixture of both conditions will guarantee optimal production.
Line 367, reference format error.
Contamination by Aspergillus cannot be avoided by better selection of waste, better decontamination, since it does not seem correct to me to present fermentation data with contaminations that can compete for substrate, produce growth inhibitors or other factors that influence fermentation.
Author Response
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Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsThis study demonstrates the potential of using a packed-bed bioreactor for solid-state fermentation of Beauveria bassiana to produce conidia, successfully scaling up from 0.5 L to 22 L with beer draff as the optimal substrate. The research identifies air-filled porosity as a crucial factor for effective fungal conidia production and shows that the packed-bed bioreactor outperforms tray bioreactors in conidia yield. The results in this manuscript are interesting, and a few comments should be addressed.
1. line 28 Add a definite article “The” before “circular bioeconomy (CBE)” since it introduces a specific concept.
2. line 30 “Sustainable Developed Goals (SDG)” should be corrected to “Sustainable Development Goals (SDGs)” as the formal term used by the United Nations is “Sustainable Development Goals,” and “Goals” is plural.
3. line 31-32 Instead of “SDG6 (sustainable management of water),” consider “SDG6 (sustainable water management)” for parallelism with other SDGs listed.
4. line 36 “from waste to resource” for more precise wording.
5. line 43 “an exponential increasing trend” should be changed to “an exponentially increasing trend”.
6. line 55-56 “SSF fermentation”change to “SSF”.
Here are some grammatical errors; please double-check the rest for any others.
7. While the article mentions the importance of CBE and SSF, it lacks sufficient background information on the significance of B. bassiana and the reasons for choosing this particular strain.
8. The article does not clearly state the specific objectives of the research. It should explicitly outline that the study aims to optimize the SSF process and scale it up for enhanced conidia production of B. bassiana.
9. The rationale for selecting specific parameters, such as air flow (AF) and the range of C/N ratios, is not clearly explained in the methodology. Please indicate whether these parameters are based on previous studies, preliminary experiments, or other sources.
10. Please use a three-line table for table 2.
11. Please realign the layout of the horizontal axis in Figure 4.
12. The discussion section mentions the significance of the experimental results but does not provide a deep comparison with other studies. The practical implications of the results are also not fully discussed.
13. Line 367 There is an error in the brackets of citation [22].
14. There are significant formatting issues throughout the text. It is recommended that the text be carefully reviewed and revised.
Comments on the Quality of English LanguageThe detains are in the comments.
Author Response
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Author Response File: Author Response.docx
Reviewer 4 Report
Comments and Suggestions for AuthorsThe manuscript describes the optimization of the production of Beauveria bassiana conidia using Solid-State Fermentation bioprocess trough packed-bed bioreactor and tray bioreactor. The authors found promising results using 22-L packed-bed reactor. As correctly highlighted by authors, the scale-up of the SSF bioprocess for an effective industrial use, still a challenge. This investigation contributes to overcome this gap. The document is well written. However, I have some observation before its acceptance.
1. The SSF bioprocess presents advantages and disadvantages. Some advantages were mentioned by authors, but others need to be included. For example, the similarity with natural conditions found by microorganisms, reduction of the possibility of the inhibition by substrate or products, among others.
2. Considering that different raw materials were used in the study, information on the particle size of each one is important. The granulometry of the particles impacts on the substrate compression and, consequently, on other fermentative parameters, including the necessity or not of the use of mixtures. The AFP is affected. Also include information on moistening of the substrates. Which is the initial humidity condition for each substrate? Discussion on the maintenance and variation of the moistening must be included.
3. Figure 1. What is the mean of the number “8” indicated in the left side of the tray reactor design? In addition, it was used 1.5 kg of substrate (500 per tray) in tray bioreactor. Despite this, it is necessary to specify the thickness of the substrate layer for each tray. This aspect can affect the microbial development (for example, the hyphae penetration index; internal temperature of the layer…).
4. Table 2 presents the DoE 1 and DoE 2. The MC, IC and AF were considered for the former and T, C/N and MC for the second. Why the option was to perform these analyzes separated? A Plackett-Burman design would be used considering these parameters simultaneously. In addition, it was said that the best results were obtained with reactor 6 and reactor 9, but the results were not shown. Please, add information on the results obtained for these two conditions.
5. Figure 3. Add information on “BB” and “AN” means in the figure caption.
6. Line 178. For the centrifugation parameter, the g force should be used and not the rpm. The same rpm values used in different equipment does not correspond to the same centrifugation condition.
7. Line 337. Replace “3ºC” by “3ºC”.
Comments on the Quality of English LanguageMinor issues.
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThank you very much for your comments and observations, however, regarding the optimization process it is not clear, they do not present any optimization graph such as a response surface graph, Pareto graph or ANOVA to see if the design is well done, well adjusted if the correlation coefficient of the data is high or low. None of the above is presented, and even if they mention that it was done as another author did, it is necessary that they present it in their work.
The authors add the following information in lines 276 and 277 “Despite using optimal production conditions (70% moisture, 6.5x106 conidia ml-1 inoculum concentration, 20 ml min-1 airflow, 25 ℃ temperature and 40 C/N ratio) [12],”
1. Where and how did they obtain these results, because in point 2.7 they do not mention what analysis software they used to model and they have to demonstrate that they really obtained this data.
2. The values ​​obtained are integers, 70%, 20 mL, 25 C and out there, when a mathematical model is used when a maximum value is obtained and very rarely are they integer values, and if they have rounded it, then they are doing it wrong, because they were optimized and they obtain an optimal temperature value of 25.4 and they use 25 or a C/N ratio of 39.6 and they use 40, so why optimize??
3. These values ​​must be demonstrated by the mathematical model, and present the same.
Author Response
Please see attached file
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsA few comment, such as the strain selection basis is not given. This is very essential. Besides, the discussion is not further improved.
Comments on the Quality of English LanguageThe authors still not revised the manuscript carefully.
Author Response
Please see attached files
Author Response File: Author Response.docx
