Strategies for Supplying Precursors to Enhance the Production of Secondary Metabolites in Solid-State Fermentation

Round 1

Reviewer 1 Report

The manuscript entitled “Strategies for supplying precursors to enhance production of secondary metabolites in solid-state fermentation “ provides a comprehensive and detailed exploration of the main biosynthetic pathways of secondary metabolism and the strategies employed to enhance secondary metabolite production in  solid-state fermentation  through precursor supplementation. The information presented is well-organized and effectively conveys the complex biochemical processes involved in these pathways. Here are some detailed comments and suggestions:

1.     Consider incorporating visual aids, such as figures or diagrams, to illustrate the key stages within the biosynthetic pathways. The inclusion of flowcharts or diagrams could significantly aid readers in comprehending the sequential reactions and transformations that occur.

2.     It would be beneficial to establish a concise link between the section discussing biosynthetic pathways and the central theme of your review paper, the enhancement of production through precursor supplementation. This connection will serve as a reminder to readers of the overarching context and purpose of the section.

3.     I recommend briefly contrasting fungal and bacterial pathways to highlight both their commonalities and divergences in terms of biosynthetic mechanisms and potential applications. This comparison could offer valuable insights into the versatility of the strategies discussed.

4.     Provide more comprehensive details on how the introduction of precursors influences specific metabolic pathways or enzymatic reactions, resulting in the observed increase in product formation. A deeper mechanistic understanding of this process would elevate the depth and quality of the ensuing discussion.

5.     Address potential limitations or challenges associated with precursor supply strategies.

6.     Discuss factors such as substrate inhibition, metabolic redirection, or any other constraints that might impact the broader applicability and success of your proposed approaches.

I have provided more comments on the attached file, please check and revise.

 

Comments for author File: Comments.pdf

Author Response

We appreciate the reviewer' helpful suggestions. All the line numbers cited below refer to those in the corrected manuscript. Changes in the corrected manuscript are highlighted in yellow when they are modifications and in green when they are additions.

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REVIEWER 1, COMMENT 1. Consider incorporating visual aids, such as figures or diagrams, to illustrate the key stages within the biosynthetic pathways. The inclusion of flowcharts or diagrams could significantly aid readers in comprehending the sequential reactions and transformations that occur.

OUR COMMENTS. We agree.

MODIFICATIONS.

1. It was introduced the Fig. 1 (Line 126).
2. The text “Primary and secondary metabolic pathways are interrelated, as shown in Figure 1. The regulation of secondary metabolism is a complex process. Therefore, in order to improve the production of secondary metabolites by microorganisms, it is essential to understand the metabolic pathways involved.” was introduced (Line 120 - 123).

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REVIEWER 1, COMMENT 2. It would be beneficial to establish a concise link between the section discussing biosynthetic pathways and the central theme of your review paper, the enhancement of production through precursor supplementation. This connection will serve as a reminder to readers of the overarching context and purpose of the section.

OUR COMMENT. We consider that sections link well. Then, new text only would be redundant.

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REVIEWER 1, COMMENT 3. I recommend briefly contrasting fungal and bacterial pathways to highlight both their commonalities and divergences in terms of biosynthetic mechanisms and potential applications. This comparison could offer valuable insights into the versatility of the strategies discussed.

MODIFICATIONS.

1. The following paragraphs were added to lines 198 – 226.

“Finally, it should be emphasized that although common molecular patterns and principles underlie life's diverse forms, many differences in primary biosynthetic path-ways exist. Many biosynthetic pathways are specific to certain groups of organisms. For instance, pheammonium lyase is a ubiquitous enzyme in fungi that catalyzes the deamination of L-Phe to trans-cinnamic acid. Conversely, only a few cinnamic and benzoic acid-derived metabolites have been described in prokaryotes [66].

From a practical perspective, the choice between using fungi or bacteria depends on the specific metabolite to be produced. Particular fungal or bacterial species exclusively synthesize some metabolites. Vancomycin, a glycopeptide antibiotic class, is produced by Amycolatopsis (formerly Streptomyces) species, such as A. orientalis or A. keratiniphila [67,68]. Although vancomycin is effective against methicillin-resistant Staphylococcus aureus infections [68], the emergence of vancomycin-resistant S. aureus has prompted the development of second-generation glycopeptide antibiotics. An example is the recently FDA-approved compound oritavancin. Although semi-synthetic, its production still relies on the in vivo production of vancomycin by Amycolatopsis species, and then, this chassis is modified by incorporating a 4-(4-chlorophenyl) benzyl group through reductive alkylation [68].

Similarly, some secondary metabolites are exclusively synthesized by fungi. Beauvericin belongs to the cyclic hexadepsipeptide family and is produced via a non-ribosomal pathway utilizing beauvericin synthetase. It sequentially binds hydroxy isovaleric acid and N-methyl-phenylalanine molecules [69]. Certain entomopathogenic fungi generate Beauvericin, which exhibits diverse biological activities, including insecticidal, antimicrobial, and antitumor properties. Due to the intricate nature of its chemical synthesis, beauvericin's production is predominantly accomplished via in vivo biosynthesis using specialized producer strains. Recently, Vásquez-Bonilla et al. [69] reported an enhancement in beauvericin production using solid-state cultures of Fusarium oxysporum AB2 com-pared to liquid cultures, increasing the yield from 0.8 mg/L to 65.3 mg/L. Moreover, they further improved yields by employing mixed cultures of F. oxysporum AB2 and Epicoccum nigrum TORT, producing 84.6 mg/L.”

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REVIEWER 1, COMMENT 4. Provide more comprehensive details on how the introduction of precursors influences specific metabolic pathways or enzymatic reactions, resulting in the observed increase in product formation. A deeper mechanistic understanding of this process would elevate the depth and quality of the ensuing discussion.

OUR COMMENT. We consider convenient to unite our answers to this comment, the comment 5 of the Reviewer 1, and comments 20 and 21 of the Reviewer 3.

MODIFICATION.

1. The following paragraphs were added to lines 534 – 572.

“Biological or physical factors may limit bioreactor performance. Considering the bio-conversion of exogenous precursors, enzyme activity and cell-membrane transport are the main biological factors influencing the bioreactor performance. Conversely, the adequate supply of the precursor to the cell depends on the physical proper functioning of the bio-reactor.

Exogenous precursors are molecules that break into the metabolism of a functional cell and must be assimilated by the appropriate metabolic pathway. Likewise, it is known that enzyme activity depends on substrate concentration, among other factors. Then, pre-cursor supply allows an increased substrate availability which could increase the corresponding pathway's metabolic flux, resulting in increased product formation. Nevertheless, the precursor addition effectivity to enhance product formation is subjected to constraints. The greater substrate availability increases the possibility of a substrate-enzyme face improving the enzyme reaction rate. Still, it cannot change the maximum enzyme re-action rate, an intrinsic system characteristic. Something similar has been argued by Oreb [101] for the artificial protein complexes design to eliminate metabolic bottlenecks.

On the other hand, getting the precursor to the cell is the job of the bioreaction system used. A bioreactor is a physical place that provides the conditions for cell growth and metabolite production. An excellent review of the general types of SSF bioreactors was pro-vided by Ge et al. [102]. They describe design, heat, and mass transfer for a tray, packed-bed, rotating-drum, stirred-drum, fluidized-bed, rocking-drum, and stirred-aerated bioreactors. Complementary, Chen [103] reviewed significant biological and engineering aspects of the SSF, such as water evaporation, heat transfer in porous media, the moisture relevance, evaporation heat removal, particle size and shape, and changes in solid mate-rial properties caused by microbial growth, among others. Chen [103] and Ge et al. [102] agree that scale-up is the biggest challenge or bottleneck for the industrial application of SSF. Ge et al. [102] identified heat transfer as the main problem. They argued that the rea-son is the solid material's poor thermal conductivity and heat capacity compared to a liquid medium. Chen [103] considered that heat, mass transfer, and aseptic operation are the main factors affecting the scale-up of SSF bioreactors.

If a precursor solution is sprayed on a culture of a filamentous fungus that suffers damage if the solid material is mixed, a tray bioreactor with thin beds of solid material may be a good choice. Still, in a similar case but growing yeast, a stirred-drum seems a better option since it allows enhanced heat and mass transfer, benefiting the medium homogeneity and bioconversion yield. In contrast, a packed-bed bioreactor is the best option for a volatile precursor added using the airstream to provide oxygen. However, the homogeneous distribution of the precursor can be complex due to the formation of preferential flow channels, which occur primarily when filamentous fungi are cultivated. Thus, it is clear that the appropriate selection of the bioreactor design to produce secondary metabolites successfully depends on a careful and depth analysis of each particular system.”

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REVIEWER 1, COMMENT 5. Address potential limitations or challenges associated with precursor supply strategies.

OUR COMMENT. We consider that modifications introduced as answer to the Comment 4 also attended this comment.

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REVIEWER 1, COMMENT 6. Discuss factors such as substrate inhibition, metabolic redirection, or any other constraints that might impact the broader applicability and success of your proposed approaches.

MODIFICATION.

1. The following paragraph was added to lines 527 – 533.

“The precursors are added to be transformed in a particular metabolic pathway. However, they can be substrates of enzymes that participate in more than one metabolic pathway or be converted by enzymes of low specificity. It can cause at least a part of the added precursor to be redirected to a part of the metabolism other than the intended one. The precursor or one of its derivatives must react with some key metabolites, such as acetyl-CoA or pyruvate, used in various metabolic pathways. This type of situation can cause low efficiency in using the added precursor.”

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REVIEWER 1, COMMENT 7. I have provided more comments on the attached file, please check and revise.

OUR COMMENTS.

1. The sentence we wrote (“In the case of plant secondary metabolites, it also contributes to preserving plant biodiver-sity [3].”) was too concise and its importance is not appreciated in the context of the manuscript. So, we rewrote the sentence and hope that the reviewer can now appreciate that it is indeed valuable in the context of the manuscript.

MODIFICATIONS.

1. The text “In the case of plant secondary metabolites, it also contributes to preserving plant biodiver-sity [3].” was replaced by the text “Moreover, microbial production also contributes to preserving plant biodiversity [3] since microorganisms can produce some plant secondary metabolites through the biotransfor-mation of precursors. Microbial production then allows humans to avoid the disad-vantages of plant extraction, like low yield, which leads to plant over-exploitation.” (Line 33-37).
2. The head of the first column of the Table 1 was changed from “Metabolite” to “Metabolite and its potential application”.
3. Table 1 was re-organized.
4. The text “This behavior was further observed in SSF, where the gradual supply of isoamyl alcohol to solid-state cultures of Pichia fermentans also increased the production of isoamyl acetate more than 12-fold in comparison to the basal yield obtained without adding the precursor [10].” was replaced by the text “This behavior was further observed in SSF, where the gradual supply of isoamyl alcohol to solid-state cultures of Pichia fermentans maintained a low yeast exposure to the precursor, avoiding or minimizing precursor toxicity [10]. It increased the production of isoamyl ace-tate more than 12-fold compared to the basal yield obtained without adding the precursor.” (Line 275-280).
5. It was revised throughout the manuscript that the scientific names are in italics.

 

Reviewer 2 Report

This manuscript is a review of enhancing the production of secondary metabolites through solid-state fermentation.

The manuscript is written in an understandable manner, is well-referenced, and the topic is of interest to the readers.

However, the authors need to do a complete review of the manuscript to correct errors, especially when writing scientific names of microorganisms, which should be in italics.

 

Line 251 Aspergillus ochraceous in italics.

References 19, 25, 26 41, 42. 48, 53, 69, 76, 78, 83, etc., check the italics of the microorganisms' name.

 

Line 473 It should be Fig. 2A

The language is understandable, the manuscript is well-written.

Author Response

We appreciate the reviewer' helpful suggestions. All the line numbers cited below refer to those in the corrected manuscript. Changes in the corrected manuscript are highlighted in yellow when they are modifications and in green when they are additions.

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REVIEWER 2, COMMENT 1. The authors need to do a complete review of the manuscript to correct errors, especially when writing scientific names of microorganisms, which should be in italics.

OUR COMMENT. It was revised throughout the manuscript that the scientific names are in italics.

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REVIEWER 2, COMMENT 2. Line 251 Aspergillus ochraceous in italics.

OUR COMMENT. It was corrected.

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REVIEWER 2, COMMENT 3. References 19, 25, 26 41, 42. 48, 53, 69, 76, 78, 83, etc., check the italics of the microorganisms' name.

OUR COMMENT. It was corrected.

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REVIEWER 2, COMMENT 4. Line 473 It should be Fig. 2A

OUR COMMENT. It was corrected.

Reviewer 3 Report

The review entitled «Strategies for supplying precursors to enhance production of secondary metabolites in solid-state fermentation «was evaluated.

However, the idea of such research is interesting, based on my assessment, the current did not meet the requested criteria to be published in Fermentation.

1.     I would recommend using English service or at least consultant with an English expert.

2.     The design of study is simple

Introduction

3.     L35, 45, authors should develop why some factors with impact on the Production of microbial secondary metabolites. I think that is better to develop a subsection and insert: Factors as 1.  Bioreactors, 2. Temperature, 3. Inoculation and kind of microorganisms, 4. Moisture and aw, 5. pH, 6. substrate, …)

4.     L45, what is the practical application of SSF in food industry? How about the nutritional value improvement

5.     Authors should introduce the limit of SSF, if all parameters are controlled or not, if some toxic products can be produced in the process of fermentation, ….?

6.     In the table 1, some information was missed, as the inoculum seize, the production yield, the strain, …

7.     L61, when describing Fungal pigments, authors should introduce their biological activities, stability, toxicity,

8.     The same remarks for antibiotics, a special focus should be given on Streptomyces

9.     The authors of this study should stress the novelty of this work , and some parts should be well developed an written

10.  I invite authors to add a subsection after the introduction “ Search strategy” which authors described the methodology followed in this review

The section “Main biosynthetic pathways of secondary metabolism”

11.  In this sub section, authors should describe carefully the strategies associating a biosynthetic gene cluster with the secondary metabolite it produces, it is crucial! In this regard, I invite authors to develop

1.     Strategies activating transcriptionally silent gene clusters

2.     Strategies for transcriptionally active gene clusters

At this level, a comparison should be proposed (advantages and disadvantages, …)

12.  In the part 2.1. Shikimate pathway, authors should discuss the possibility of Shikimate pathway transcriptionally on human gut microbiome

13.  L137, I invite authors to draw a good figure schematize all described pathways for this section

14.  L143. In the subsection ‘Malonic acid pathway” Authors should discuss the designing and for malonic acid production biologically

15.  L168 sub section “Using precursors to enhance secondary metabolites production, L196, authors should develop some studies on the Optimization of the culture conditions, please cite some works describing some mathematical optimization, (Statistical versus and artificial intelligence can be developed)

16.  Authors should link these two parts: “Main biosynthetic pathways of secondary metabolism » and “precursors to enhance secondary metabolites production”

17.  Authors should discuss the levels (or concentrations) of used precursors,

18.  How about the inorganic compounds and their impact on secondary metabolites production enhancement

19.  In the sub section, 4. “Enhancing secondary metabolites production in SSF” Authors should discuss: the Effect of Factor on the production of secondary metabolites under solid state fermentation: (e. g. Selection of substrate and supplements; Particle size (the ratio of surface area-to-volume); pH, Effect of sterilization;

20.  How about the Challenges for the production of SMs via SSF? In this part, I invite authors to discuss the (1) Scale-up, (2) Heat Dissipation and Mass transfer; (3) Downstream processing, …

21.  The discussion part is week, I think it is better to discuss the Aspects of design of bioreactor for the production of SMs in SSF and Overcoming limitations; as (Tray fermenters for SSF); the Rolling bed fermenters for SSF:; Continuous stirred tank reactor (CSTR):, Continuous stirred tank reactor (CSTR):, ….

22.  The conclusion should be improved taken into all my previous remarks

 

1I would recommend using English service or at least consultant with an English expert.

Author Response

We appreciate the reviewer' helpful suggestions. All the line numbers cited below refer to those in the corrected manuscript. Changes in the corrected manuscript are highlighted in yellow when they are modifications and in green when they are additions.

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REVIEWER 3, COMMENT 1. I would recommend using English service or at least consultant with an English expert.

OUR COMMENT. English was checked throughout the entire manuscript..

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REVIEWER 3, COMMENT 2. The design of study is simple

OUR COMMENT. None.

MODIFICATION. None.

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REVIEWER 3, COMMENT 3. L35, 45, authors should develop why some factors with impact on the Production of microbial secondary metabolites. I think that is better to develop a subsection and insert: Factors as 1.  Bioreactors, 2. Temperature, 3. Inoculation and kind of microorganisms, 4. Moisture and aw, 5. pH, 6. substrate, …)

OUR COMMENT. Respectfully, the introductory section should state in a general way broad aspects related to the central theme of the work. We do not detract from the reviewer's point of view, but his proposal would unnecessarily extend the manuscript's introduction.

MODIFICATION. None.

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REVIEWER 3, COMMENT 4. L45, what is the practical application of SSF in food industry? How about the nutritional value improvement

OUR COMMENT. Again, with respect, we consider that it is a situation similar to that of Comment 3.

MODIFICATION. None.

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REVIEWER 3, COMMENT 5. Authors should introduce the limit of SSF, if all parameters are controlled or not, if some toxic products can be produced in the process of fermentation, ….?

OUR COMMENT. Again, with respect, we consider that it is a situation similar to that of Comment 3.

MODIFICATION. None.

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REVIEWER 3, COMMENT 6. In the table 1, some information was missed, as the inoculum seize, the production yield, the strain, …

OUR COMMENT. The most complete information was the yields.

MODIFICATION. A column with yields was added to Table 1.

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REVIEWER 3, COMMENT 7. L61, when describing Fungal pigments, authors should introduce their biological activities, stability, toxicity,

OUR COMMENT. Again, with respect, we consider that it is a situation similar to that of Comment 3.

MODIFICATION. None.

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REVIEWER 3, COMMENT 8. The same remarks for antibiotics, a special focus should be given on Streptomyces

OUR COMMENT. Again, with respect, we consider that it is a situation similar to that of Comment 3.

MODIFICATION. None.

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REVIEWER 3, COMMENT 9. The authors of this study should stress the novelty of this work , and some parts should be well developed an written

OUR COMMENT. We regret to have little agreement with Reviewer 3. However, we believe it is because we have different concepts of the content that a review like the one we are presenting should have.

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REVIEWER 3, COMMENT 10. I invite authors to add a subsection after the introduction “ Search strategy” which authors described the methodology followed in this review

OUR COMMENT. Once again, we respectfully disagree with the comment since a search for review articles in various journals reveals that the vast majority do not contain a section on materials and methods or similar. We couldn't find a single one in Fermentation. However, we can comment here what was the methodology we followed.

 

The bibliographic searches were done in different databases (Scopus, Google Scholar, Clarivate, and PubMed). The search period was from 1990 to date, but trying to focus on documents from the last 5 to 8 years. The following keywords were used, individually or in various combinations: (1) solid-state fermentation, (2) secondary metabolite, (3) metabolic pathway, (4) engineering, (5) precursor, and (6) bioreactor.

 

The information analysis began with its classification according to the sections of the manuscript and the selection of the relevant articles. Decisions about the pieces to be included were made in discussion sessions via videoconference. The information analysis shows that the data available for SSF are scarce compared to submerged Fermentation, but it is an active growth area.

MODIFICATION. None.

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REVIEWER 3, COMMENT 11. In this sub section, authors should describe carefully the strategies associating a biosynthetic gene cluster with the secondary metabolite it produces, it is crucial! In this regard, I invite authors to develop. 1. Strategies activating transcriptionally silent gene clusters, 2. Strategies for transcriptionally active gene clusters. At this level, a comparison should be proposed (advantages and disadvantages, …)

OUR COMMENT. These issues are addressed in section 4. Enhancing secondary metabolites production in SSF.

MODIFICATION. None.

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REVIEWER 3, COMMENT 12. In the part 2.1. Shikimate pathway, authors should discuss the possibility of Shikimate pathway transcriptionally on human gut microbiome

OUR COMMENT. We consider the human gut microbiome to be a topic outside the scope of the manuscript.

MODIFICATION. None.

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 REVIEWER 3, COMMENT 13. L137, I invite authors to draw a good figure schematize all described pathways for this section

OUR COMMENT. We added a figure to schematize the described pathways.

MODIFICATION.

1. It was introduced the Fig. 1 (Line 126).
2. The text “Primary and secondary metabolic pathways are interrelated, as shown in Figure 1. The regulation of secondary metabolism is a complex process. Therefore, in order to improve the production of secondary metabolites by microorganisms, it is essential to understand the metabolic pathways involved.” was introduced (Line 120 - 123).

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REVIEWER 3, COMMENT 14. L143. In the subsection ‘Malonic acid pathway” Authors should discuss the designing and for malonic acid production biologically

OUR COMMENT. We already do what the reviewer recommends. It should be noted that this is still a support section, which is not intended to be an exhaustive and in-depth review of metabolic pathways.

MODIFICATION. None.

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REVIEWER 3, COMMENT 15. L168 sub section “Using precursors to enhance secondary metabolites production, L196, authors should develop some studies on the Optimization of the culture conditions, please cite some works describing some mathematical optimization, (Statistical versus and artificial intelligence can be developed)

OUR COMMENT. We already do what the reviewer recommends.

MODIFICATION. None.

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REVIEWER 3, COMMENT 16. Authors should link these two parts: “Main biosynthetic pathways of secondary metabolism » and “precursors to enhance secondary metabolites production”

OUR COMMENT. We consider that sections link well. Then, new text only would be redundant.

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REVIEWER 3, COMMENT 17. Authors should discuss the levels (or concentrations) of used precursors,

OUR COMMENT. We already do what the reviewer recommends. It is included in the examples that mention this information.

MODIFICATION. None.

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REVIEWER 3, COMMENT 18. How about the inorganic compounds and their impact on secondary metabolites production enhancement

OUR COMMENT. We already do what the reviewer recommends.

MODIFICATION. None.

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REVIEWER 3, COMMENT 19. In the sub section, 4. “Enhancing secondary metabolites production in SSF” Authors should discuss: the Effect of Factor on the production of secondary metabolites under solid state fermentation: (e. g. Selection of substrate and supplements; Particle size (the ratio of surface area-to-volume); pH, Effect of sterilization;

OUR COMMENT. We already do what the reviewer recommends. The section includes and discusses the information provided by the references.

MODIFICATION. None.

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REVIEWER 3, COMMENT 20. How about the Challenges for the production of SMs via SSF? In this part, I invite authors to discuss the (1) Scale-up, (2) Heat Dissipation and Mass transfer; (3) Downstream processing, …

OUR COMMENT. We consider convenient to unite our answers to this comment, the comment 21 (the next), and the comment 5 of the Reviewer 1.

MODIFICATION.

1. The following paragraphs were added to lines 534 – 572.

“Biological or physical factors may limit bioreactor performance. Considering the bio-conversion of exogenous precursors, enzyme activity and cell-membrane transport are the main biological factors influencing the bioreactor performance. Conversely, the adequate supply of the precursor to the cell depends on the physical proper functioning of the bio-reactor.

Exogenous precursors are molecules that break into the metabolism of a functional cell and must be assimilated by the appropriate metabolic pathway. Likewise, it is known that enzyme activity depends on substrate concentration, among other factors. Then, pre-cursor supply allows an increased substrate availability which could increase the corresponding pathway's metabolic flux, resulting in increased product formation. Nevertheless, the precursor addition effectivity to enhance product formation is subjected to constraints. The greater substrate availability increases the possibility of a substrate-enzyme face improving the enzyme reaction rate. Still, it cannot change the maximum enzyme re-action rate, an intrinsic system characteristic. Something similar has been argued by Oreb [101] for the artificial protein complexes design to eliminate metabolic bottlenecks.

On the other hand, getting the precursor to the cell is the job of the bioreaction system used. A bioreactor is a physical place that provides the conditions for cell growth and metabolite production. An excellent review of the general types of SSF bioreactors was pro-vided by Ge et al. [102]. They describe design, heat, and mass transfer for a tray, packed-bed, rotating-drum, stirred-drum, fluidized-bed, rocking-drum, and stirred-aerated bioreactors. Complementary, Chen [103] reviewed significant biological and engineering aspects of the SSF, such as water evaporation, heat transfer in porous media, the moisture relevance, evaporation heat removal, particle size and shape, and changes in solid mate-rial properties caused by microbial growth, among others. Chen [103] and Ge et al. [102] agree that scale-up is the biggest challenge or bottleneck for the industrial application of SSF. Ge et al. [102] identified heat transfer as the main problem. They argued that the rea-son is the solid material's poor thermal conductivity and heat capacity compared to a liquid medium. Chen [103] considered that heat, mass transfer, and aseptic operation are the main factors affecting the scale-up of SSF bioreactors.

If a precursor solution is sprayed on a culture of a filamentous fungus that suffers damage if the solid material is mixed, a tray bioreactor with thin beds of solid material may be a good choice. Still, in a similar case but growing yeast, a stirred-drum seems a better option since it allows enhanced heat and mass transfer, benefiting the medium homogeneity and bioconversion yield. In contrast, a packed-bed bioreactor is the best option for a volatile precursor added using the airstream to provide oxygen. However, the homogeneous distribution of the precursor can be complex due to the formation of preferential flow channels, which occur primarily when filamentous fungi are cultivated. Thus, it is clear that the appropriate selection of the bioreactor design to produce secondary metabolites successfully depends on a careful and depth analysis of each particular system.”

 **************************************

REVIEWER 3, COMMENT 21. The discussion part is week, I think it is better to discuss the Aspects of design of bioreactor for the production of SMs in SSF and Overcoming limitations; as (Tray fermenters for SSF); the Rolling bed fermenters for SSF:; Continuous stirred tank reactor (CSTR):, Continuous stirred tank reactor (CSTR):, ….

OUR COMMENT. Please see the previous comment.

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REVIEWER 3, COMMENT 22. The conclusion should be improved taken into all my previous remarks

OUR COMMENT. We agree.

MODIFICATION.

1. The sentence “Appropriate selection of the type of bioreactor is also a transcendental factor to reach a high precursor bioconversion.” was added to lines 587 – 588.
2. The sentence “Finally, more rapid progress must be made in scaling up SSF bioreactors to achieve signif-icant participation of this technology in the commercial production of microbial second-ary metabolites.” was added to lines 591 – 593.

 

Round 2

Reviewer 3 Report

accept