Study on the Operational Modes Using Both Growing and Resting Cells for Succinic Acid Production from Xylose Kinetic Modelling
Round 1
Reviewer 1 Report
This work gives a bioprocess for SA production using a synthetic growth medium and xylose as carbon source has been demonstrated. Some experimental results have been obtained, which has certain significance. But serious changes are needed in terms of innovative validation and language. Thus, this manuscript should be make revision.
1.How to explain the innovation of this work is a question worthy of attention, please make corresponding modifications.
2. Authors said: "This article verifies, for the first time, the possibility of producing succinic acid with fed-batch or repeated batch operations with Actinobacillus succinogenes in a resting state, that is, in the absence of a nitrogen source. " So, why？ What is the significance of no nitrogen source for industrial applications?
3. Please change the language throughout. The language is too colloquial.
Author Response
Manuscript´s reference: Fermentation 2482909
Title: Study on the operational modes using both growing and resting cells for the succinic acid production from xylose. Kinetic modelling
Journal: Fermentation
We are grateful to the reviewer for their careful revision and comments. They have aided us to improve the original manuscript. The whole manuscript has been revised having into account all the comments from the reviewers. Changes in the document have been highlighted using coloured red font. We describe bellow a list of changes and/or a rebuttal against each point which has been included in the revised manuscript.
Reviewer 1
(I) Please check that all references are relevant to the contents of the manuscript.
After reviewing the bibliographical references, it has been considered that all of them are necessary for an appropriate understanding and justification of the article.
Reviewer 1
This work gives a bioprocess for SA production using a synthetic growth medium and xylose as carbon source has been demonstrated. Some experimental results have been obtained, which has certain significance. But serious changes are needed in terms of innovative validation and language. Thus, this manuscript should be make revision.
Comment 1. How to explain the innovation of this work is a question worthy of attention, please make corresponding modifications.
Thank you very much for your suggestion, clarifications on the innovation of this work have been included in the following sections:
-abstract:
“This article verifies, for the first time, the possibility of producing succinic acid with fed-batch or repeated batch operations with Actinobacillus succinogenes in a resting state, that is, in the absence of a nitrogen source. In this work it is possible to optimize separately the stages of cell growth and production in fed-batch or repeated batch, minimizing the costs associated with the nitrogen source and facilitating the subsequent purification of SA.”
-introduction
“In this present study, SA production using A. succinogenes in both growth and resting states has been researched, exploring different forms of operation through biocatalyst re-use by means of
fed-batch and repeated batch. This is the first work in which the operation with resting cells is studied in different types of operation, allowing to optimize the stages of cell growth and SA production separately and reduces the operating costs associated with the nitrogen source and SA purification.”
-conclusions
“This means that, for the first time, it has been possible to produce succinic acid during three cycles of xylose feeding in the absence of a nitrogen source from a single stage of cell growth, also achieving yields and selectivities superior to the equivalent operation with growing cells, in other words, in a culture medium with nitrogen source.”
Comment 2. Authors said: "This article verifies, for the first time, the possibility of producing succinic acid with fed-batch or repeated batch operations with Actinobacillus succinogenes in a resting state, that is, in the absence of a nitrogen source." So, why? What is the significance of no nitrogen source for industrial applications?
The purpose of using resting cells is to separate the growth and production processes to see if the established submerged culture procedure can be improved. In fact, this strategy allows for the separate optimization of inoculum and acid production stages. The experience of the research group in the development of this type of process has shown that it is generally a good solution.
In order to obtain cells for subsequent use in the production stage, it is necessary to carry out a conventional fermentation, although it requires the optimization of conditions so that the cells have the appropriate metabolic state for their subsequent use in resting cells. A notable advantage of separating the stages of growth (or obtaining the biocatalyst) and production (with the cells in the resting cell state) is the culture medium needed in the stages. Generally, to carry out production with growing cells, culture media with components such as yeast extract, etc. are used, which, although they do not have a high concentration in the medium, if the total mass is calculated to carry out the process in industrial bioreactors, would entail a high price. When the biomass is produced, it is done on a much smaller scale, so the need for such culture media containing expensive nitrogen sources is considerably reduced. However, the medium used for resting cell production is very simple and, therefore, cost-effective.
Separating growth and production into two different steps allows a better optimization of each step and of the overall process, as mentioned before. Considering the scale-up, the proposed bioprocess would lead to higher productivity linked to the reduction of dead times as well as to avoid substrate inhibition that was observed when using growing cells as biocatalyst. In addition, this separation allows reducing the formation of by-products, which is especially critical in the case of SA production since 60% of its costs correspond to the purification stage.
This information has been included in the first paragraph of page 3.
“Operating under nitrogen limited conditions, and formulating a medium composed exclusively of a carbon source and a buffer solution to maintain osmotic pressure, resting cells are maintained metabolically active even though cell growth is impeded. In this way, the metabolic pathways are active towards SA production and cellular maintenance but there is no bacterial growth. Concurrently, the number of by-products is also reduced dramatically [18-20], this being one of the main advantages associated with this type of operation. In fact, Escanciano et al. (2022) [17] managed to reduce the quantity of by-products generated by 27.5% in the production of SA from xylose. In order to obtain cells for subsequent use in the production stage in resting state, it is necessary to carry out a conventional fermentation, although it requires the optimization of conditions so that the cells have the appropriate metabolic state for their subsequent use in resting cells. A notable advantage of separating the stages of growth (or obtaining the biocatalyst) and production (with the cells in the resting cell state) is the culture medium needed in the stages. Generally, to carry out production with growing cells, culture media with components such as yeast extract, etc. are used, which, although they do not have a high concentration in the medium, if the total mass is calculated to carry out the process in industrial bioreactors, would entail a high price. When the biomass is produced, it is done on a much smaller scale, so the need for such culture media is considerably reduced. However, the medium used for resting cell production is very simple and cost-effective, devoid of nitrogen sources, usually expensive. Separating growth and production into two different steps allows a better optimization of each step and of the overall process. Considering the scale-up, the proposed bioprocess would lead to higher productivity linked to the reduction of dead times as well as to avoid substrate inhibition that was observed when using growing cells as biocatalyst [18,20,21].”
Comment 3. Please change the language throughout. The language is too colloquial.
The manuscript has been revised by the “Francisco de Vitoria” University Language Centre expert personnel to improve English style and correct grammar mistakes.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The work is dedicated to optimizing the process of obtaining succinic acid (SA) from xylose у Actinobacillus succinogenes. The work was done at a good methodological level, written in a clear, understandable scientific language, which facilitates the process of understanding all the nuances of A. succinogenes fermentation in order to obtain succinic acid from xylose obtained as waste from the wood processing industry. Operating under nitrogen limited conditions (so called resting state), the authors showed the fundamental possibility of optimizing the bioprocess for SA production. It is noteworthy that the authors managed to optimize the fermentation in such a way that the highest SA yield in fed batch mode were reached with resting cell, which had limited nutrients availability. The fundamental advantage (which was demonstrated in some experiments of the present work) of using resting cells for the biosynthesis of a metabolite containing only carbon, oxygen, and hydrogen atoms in its composition is important from a methodological point of view. In fact, the cellular resources of A. succinogenes that could be spent on growth are redirected to SA biosynthesis.
The authors also carry out kinetic modeling of processes, where, in addition to xylose and SA, they operate with by-products (BP). For a clearer interpretation of the results, it would be good to present schemes of the metabolic pathways for the conversion of xylose to SA, as well as present the BP formed during metabolism.
Comments
Line 52
Check if there is an extra dot in this line
Figures 1 (figure caption)
Why, according to the legend, the substrate (Xyl, marked in red) grows during the fermentation process, while the product (SA, marked in blue) falls?
It is possible that the notation for Xyl and SA is mixed up in the legend.
Figures 2 (figure caption)
Again, why, according to the legend, the substrate (Xyl, marked in red) grows during the fermentation process, while the product (SA, marked in blue) falls?
Correct notations for Xyl and SA.
Figures 3 (figure caption)
And again, why, according to the legend, the substrate (Xyl, marked in red) grows during the fermentation process, while the product (SA, marked in blue) falls?
Correct notations for Xyl and SA.
Author Response
Manuscript´s reference: Fermentation 2482909
Title: Study on the operational modes using both growing and resting cells for the succinic acid production from xylose. Kinetic modelling
Journal: Fermentation
We are grateful to the reviewer for their careful revision and comments. They have aided us to improve the original manuscript. The whole manuscript has been revised having into account all the comments from the reviewers. Changes in the document have been highlighted using coloured red font. We describe bellow a list of changes and/or a rebuttal against each point which has been included in the revised manuscript.
Reviewer 2
The work is dedicated to optimizing the process of obtaining succinic acid (SA) from xylose and Actinobacillus succinogenes. The work was done at a good methodological level, written in a clear, understandable scientific language, which facilitates the process of understanding all the nuances of A. succinogenes fermentation in order to obtain succinic acid from xylose obtained as waste from the wood processing industry. Operating under nitrogen limited conditions (so called resting state), the authors showed the fundamental possibility of optimizing the bioprocess for SA production. It is noteworthy that the authors managed to optimize the fermentation in such a way that the highest SA yield in fed batch mode were reached with resting cell, which had limited nutrients availability. The fundamental advantage (which was demonstrated in some experiments of the present work) of using resting cells for the biosynthesis of a metabolite containing only carbon, oxygen, and hydrogen atoms in its composition is important from a methodological point of view. In fact, the cellular resources of A. succinogenes that could be spent on growth are redirected to SA biosynthesis.
Comment 1. The authors also carry out kinetic modelling of processes, where, in addition to xylose and SA, they operate with by-products (BP). For a clearer interpretation of the results, it would be good to present schemes of the metabolic pathways for the conversion of xylose to SA, as well as present the BP formed during metabolism.
A figure regarding the involved metabolic pathways in succinic acid production was included in section 1. Introduction (see Figure 1). In section 3.4, a further explication relating to lumping by products i kinetic model´s consideration has been included:
“The only by-products detected in the culture medium were acetic and formic acids, which are formed almost directly from pyruvate, as is reflected in Figure 1. Moreover, the evolution of their concentrations exhibited remarkable similarity across all experiments. Based on that, Acetic and formic acids concentrations were lumped or summed to obtain a global by product concentration in the kinetic model.”
Comment 2. Line 52: Check if there is an extra dot in this line.
Punctuation marks have been carefully revised in order to correct mistakes.
Comment 3.1. Figures 1 (figure caption): Why, according to the legend, the substrate (Xyl, marked in red) grows during the fermentation process, while the product (SA, marked in blue) falls? It is possible that the notation for Xyl and SA is mixed up in the legend.
Comment 3.2. Figures 2 (figure caption): Again, why, according to the legend, the substrate (Xyl, marked in red) grows during the fermentation process, while the product (SA, marked in blue) falls? Correct notations for Xyl and SA.
Comment 3.3. Figures 3 (figure caption): And again, why, according to the legend, the substrate (Xyl, marked in red) grows during the fermentation process, while the product (SA, marked in blue) falls? Correct notations for Xyl and SA.
As the reviewer pointed out, figure caption in Figures 1, 2 and 3 were wrong in the previous manuscript. The mistake has been corrected in the revised manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
This manuscript can be accepted with present form.
