Biological Stoichiometric Analysis during Substrate Utilization and Secondary Metabolite Production by Non-Saccharomyces Yeasts Using Grape Pomace Extract as Fermentation Medium

Round 1

Reviewer 1 Report

The manuscript by Mewa-Ngongang and colleagues reports a computational/engineering analysis of three yeast strains (Candida pyralidae, Pichia kluyveri and Pichia kluyveri) growing on (fermenting?) grape pomace. Thanks to a few biochemical characterization of the fermentation products, the authors could set up simple biological stoichiometric models allowing to predict the efficiency of biopreservative production and hence identifying the most relevant factors to be considered in the design of the process.

The study seems properly designed from the mathematical and engineering viewpoint (this is not my field of expertise, though), but the biological rationale should be better defined or reformulated.

In particular, I would suggest the authors carefully consider that the stoichiometric models and bioenergetic parameters they analyzed over this study may not necessarily indicative of the potential of the yeast or of the process to produce biopreservatives. Biopreservation (and the inhibition of spoilage microorganisms as tested in the presented study) may occur thanks to the synthesis of (secondary) metabolites. Despite the models and the factors investigated in this study do allow the optimization of substrate consumption and energy acquisition, they do not necessarily indicate that the process leads to the production of the inhibitory compounds. The authors report that “the efficiency of microorganisms towards extracellular compounds production can be determined by the stoichiometric coefficients and bioenergetic analysis”, and this may be correct, but not for any type of extracellular compound, nor it can be assumed that the extracellular components are these responsible for biopreservation.

Mathematical models based on the metabolic pathways in- output are used to include this possibility, but I do understand that it may not be possible to implement such models in non-model species (e.g. non-Saccharomyces cerevisiae).

Hence, I would suggest the authors either further support the association between substrate consumption and production of the compounds promoting biopreservation or propose the study is not aimed at supporting the optimization of biopreservation production, but rather at maximizing the consumption of agro-waste material.

Furthermore, the authors should comment on the fact that the model and bioenergetic factors reported over this study may be correct for the investigated strain, but not actually representative of the entire yeast species.

Author Response

Rebuttals associated with the manuscript titled “Biological stoichiometric analysis during biopreservatives production by non-Saccharomyces yeasts using grape pomace extracts as fermentation medium.”

Manuscript ID: fermentation-1214797

Statement of appreciation

The authors would like to firstly thank the reviewers for agreeing to review the manuscript. Secondly, we thank the reviewers for their insightful and constructive feedback. It is greatly appreciated. Please find our responses below and all the sections corrected or modified have been highlighted in yellow throughout the manuscript.

Reviewer 1

The manuscript by Mewa-Ngongang and colleagues reports a computational/engineering analysis of three yeast strains (Candida pyralidae, Pichia kluyveri and Pichia kluyveri) growing on (fermenting?) grape pomace.

Thanks to a few biochemical characterization of the fermentation products, the authors could set up simple biological stoichiometric models allowing to predict the efficiency of biopreservative production and hence identifying the most relevant factors to be considered in the design of the process.

Comment 1

The study seems properly designed from the mathematical and engineering viewpoint (this is not my field of expertise, though), but the biological rationale should be better defined or reformulated.

Response

Thank you for this valuable comment, the authors agree and have reformulated the biological rationale (Please see highlighted section in the article).

Comment 2

In particular, I would suggest the authors carefully consider that the stoichiometric models and bioenergetic parameters they analyzed over this study may not necessarily indicative of the potential of the yeast or of the process to produce biopreservatives. Biopreservation (and the inhibition of spoilage microorganisms as tested in the presented study) may occur thanks to the synthesis of (secondary) metabolites.

Response

The authors agree and have made the necessary corrections; however, it is worth mentioning that the secondary metabolites are part of the products of the process, which can be accounted for in the mass balances of the stoichiometric models.

Comment 3

Despite the models and the factors investigated in this study do allow the optimization of substrate consumption and energy acquisition, they do not necessarily indicate that the process leads to the production of the inhibitory compounds. The authors report that “the efficiency of microorganisms towards extracellular compounds production can be determined by the stoichiometric coefficients and bioenergetic analysis”, and this may be correct, but not for any type of extracellular compound, nor it can be assumed that the extracellular components are these responsible for biopreservation.

Response

We acknowledge the value of this comment and find it very useful. The authors agree and have made the necessary corrections.

Comment 4

Mathematical models based on the metabolic pathways in- output are used to include this possibility, but I do understand that it may not be possible to implement such models in non-model species (e.g. non-Saccharomyces cerevisiae).Hence, I would suggest the authors either further support the association between substrate consumption and production of the compounds promoting biopreservation or propose the study is not aimed at supporting the optimization of biopreservation production, but rather at maximizing the consumption of agro-waste material.

Response

Thank you for raising this valid point, the authors agree and have made the necessary corrections.

Comment 5

Furthermore, the authors should comment on the fact that the model and bioenergetic factors reported over this study may be correct for the investigated strain, but not actually representative of the entire yeast species.

Response

The authors agree and have made the necessary corrections.

Author Response File: Author Response.pdf

Reviewer 2 Report

Result of this comprehensive work stands on its own, worth to be published. Due to the very specific area of the subject, amount of readers will be low, but that doesn't limit quality of manuscript.

It is recommended that the given results should be compared with other agro-waste from other soources like molasses from sugar beads. Should/could be subject for another publication to make things round.

Author Response

Rebuttals associated with the manuscript titled “Biological stoichiometric analysis during biopreservatives production by non-Saccharomyces yeasts using grape pomace extracts as fermentation medium.”

Manuscript ID: fermentation-1214797

Statement of appreciation

The authors would like to firstly thank the reviewers for agreeing to review the manuscript. Secondly, we thank the reviewers for their insightful and constructive feedback. It is greatly appreciated. Please find our responses below and all the sections corrected or modified have been highlighted in yellow throughout the manuscript.

Reviewer 2

Comment 1

Result of this comprehensive work stands on its own, worth to be published. Due to the very specific area of the subject, amount of readers will be low, but that doesn't limit quality of manuscript.

Response

The authors acknowledge the honest opinion about the article in relation to the area of the subject.

Comment 2

It is recommended that the given results should be compared with other agro-waste from other soources like molasses from sugar beads. Should/could be subject for another publication to make things round.

Response

The authors agree with the suggestion and have included that in the recommendation sentence of our conclusion.

Author Response File: Author Response.pdf