Enhancing Succinic Acid Production by Sequential Adaptation of Selected Basfia succiniciproducens Strains to Arundo donax Hydrolysate

Round 1

Reviewer 1 Report

The manuscript authored by Robertiello et al. presents original results in the topic of biobased succinic acid production (SA) using selected Basfia succiniciproducens bacterial strains and hydrolysate from Arundo donax (elephant grass) as renewable carbohydrate source. The main novelty of this study is the development of cell lines which can tolerate the presence of certain concentrations of organic acids (succinic, acetic, and lactic) which are the main fermentation end-products, as well as of by-products with known toxicity for the microorganism, furfural and hydroxymethyl furfural. The manuscript looks like a continuation of other studies published in the same topic, demonstrating a constant scientific interest for the utilization of Basfia succiniciproducens strains for adipic acid production. Although being of potential scientific interest and well written, I consider that it needs some adjustment before recommending it for publication.

1. Research results into the same topic, the biobased production of succinic acid using Basfia succiniciproducens and Arundo donax, were also reported in other publications coauthored by some contributors of this manuscript. Some of these works are cited, particularly in the methods part (references #13 and #14), but others, e.g., https://0-doi-org.brum.beds.ac.uk/10.1186/s13068-019-1362-6 and https://0-doi-org.brum.beds.ac.uk/10.1007/s12155-017-9814-y are not mentioned. I would like to ask the authors to comparatively discuss the actual findings related to all these previous publications, clearly specifying the advancement in the field brought by the approach developed here.  

2. I have found two recent publications in the field of biobased succinic acid production which are not mentioned in this manuscript, https://0-doi-org.brum.beds.ac.uk/10.1016/j.biortech.2021.126224, and https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation9010046. It would be useful to do it, also for comparison with the own results.

3. Please specify the carbohydrate (cellulose and hemicellulose) content of the A. donax biomass used as raw material, as well as the succinic acid yield related to the theoretically available sugar from this biomass, to allow a global view about the efficiency of the whole process.  As indicated in the abstract, about 5.8 g/L of succinic acid were the best results obtained using the adapted cell lines 2E and 4D at laboratory scale and about 17.2 g/L at pilot scale, respectively. A previously mentioned paper (Cimini et al. https://0-doi-org.brum.beds.ac.uk/10.1186/s13068-019-1362-6) claims 88.5% succinic acid production per kg of A. donax biomass, using non-adapted Basfia succiniciproducens strains. I invite the authors to comment these results.

4. Abstract, line 19: succinic acid is not by-product of the fermentation, it represents the main end-product of interest

The quality of the English is good. I have found only one minor error.

- Please italicize the Latin names in the abstract;

Author Response

1) Research results into the same topic, the biobased production of succinic acid using Basfia succiniciproducens and Arundo donax, were also reported in other publications coauthored by some contributors of this manuscript. Some of these works are cited, particularly in the methods part (references #13 and #14), but others, e.g., https://0-doi-org.brum.beds.ac.uk/10.1186/s13068-019-1362-6 and https://0-doi-org.brum.beds.ac.uk/10.1007/s12155-017-9814-y are not mentioned. I would like to ask the authors to comparatively discuss the actual findings related to all these previous publications, clearly specifying the advancement in the field brought by the approach developed here.

We sincerely appreciate your observation regarding the previous publications. We acknowledge that there are additional publications that have not been mentioned.

In response to your request, we provided a comparative discussion of the actual findings related to all the previous publications, including those you mentioned.

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2) I have found two recent publications in the field of biobased succinic acid production which are not mentioned in this manuscript, https://0-doi-org.brum.beds.ac.uk/10.1016/j.biortech.2021.126224, and https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation9010046. It would be useful to do it, also for comparison with the own results.

Thank you for bringing those two recent publications on SA production to our attention. We agree that citing these works is valuable input to improve and provide a more comprehensive overview of the field and for facilitating a comparison with our results. For these reasons, we added these references in the discussion section.

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3)        Please specify the carbohydrate (cellulose and hemicellulose) content of the A. donax biomass used as raw material, as well as the succinic acid yield related to the theoretically available sugar from this biomass, to allow a global view about the efficiency of the whole process. As indicated in the abstract, about 5.8 g/L of succinic acid were the best results obtained using the adapted cell lines 2E and 4D at laboratory scale and about 17.2 g/L at pilot scale, respectively. A previously mentioned paper (Cimini et al. https://0-doi-org.brum.beds.ac.uk/10.1186/s13068-019-1362-6) claims 88.5% succinic acid production per kg of A. donax biomass, using non-adapted Basfia succiniciproducens strains. I invite the authors to comment these results.

We appreciated your suggestion and have carefully considered it. Based on the characterizations conducted on the starting biomass {#41}, yield estimation was focused on xylans and glucans, rather than cellulose and hemicellulose.

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It is important to highlight that the results presented in our work were obtained in a laboratory scale without the implementation of a fed-batch process with addition of pure Glucose. Consequently, the observed yield values may appear significantly lower compared to the mentioned study by Cimini et al., {#31}, which reported a succinic acid production of 88.5% per kg of A. donax biomass using non-adapted Basfia succiniciproducens strains. However, as the use of adapted bacterial lines in this context is a new area of research, pilot-scale deployment or strain engineering cannot be excluded as potential future investigation. These approaches could provide valuable insights into further optimizing the process and potentially enhancing the overall yield of SA production.

4) Abstract, line 19: succinic acid is not by-product of the fermentation, it represents the main end-product of interest

Thank you for pointing out this issue in the abstract regarding the characterization of succinic acid as a by-product of fermentation. We appreciate your keen observation and thus we clarify and rectify this inaccuracy.

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Thank you once again for your time and effort in reviewing our work. We appreciated all your valuable input.

Reviewer 2 Report

The topic of the study fits within the scope of the journal, and the experimental work is organized with general methods. In general the paper looks well written, clearly described in all single sections together with the discussion of the results. But there are still few remaining questions/comments listed below in detail:

But first of all: The percentage of the cited literature in the range of “2015 or older” is >35%. From this perspective, it would be necessary to cite the current literature in order to justify more thoroughly the novelty of the present work and/or further substantiate the proposed progress. The update of the references would have impact on the sections “introduction/state of the art” and  “results and discussion”!

It would be helpful to include a table with already published figures comparing the results here obtained. I imagine there are not that many (directly) comparable papers already published, but ultimately the approach presented here has to compete with ALL other succinic production methods in terms of productivity or other performance parameters.

The use of alternative carbon sources is seen as the most promising approach in the introduction, so the question arises as to what solid fraction (section 2.5) remains after solid-liquid separation or what could be done with it? Or to put the question another way - would this residual solid matter interfere with the later fermentation? With regard to your discussion (lines 379-384), it would be interesting to know with what yield the pre-treatment and hydolysis took place or whether >20% of the biomass could also be used.

The procedure of the different screening and adaptation stages is logical and therefore the following question is not really a criticism, but in view of the relatively small differences between the strains shown in Figure 2, it could be that you would come to different results if you had already carried out the tests (section 2.5) in the bioreactor at constant pH instead of static shake flasks. Could you please comment briefly on this based on your own experience?

It is probably just an accidental wording to list SA in the summary (line 19) as a "by-product"...!?

Author Response

1) But first of all: The percentage of the cited literature in the range of “2015 or older” is >35%. From this perspective, it would be necessary to cite the current literature in order to justify more thoroughly the novelty of the present work and/or further substantiate the proposed progress. The update of the references would have impact on the sections “introduction/state of the art” and “results and discussion”!

Thank you for your valuable feedback. We took your opinion into consideration, and as a result, we integrated and modified the sections introduction/state of the art” and “results and discussion”, with additional references.

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We include more recent citations, as per your suggestion, and the percentage of citations prior to 2015 should now be below 35%.

2) It would be helpful to include a table with already published figures comparing the results here obtained. I imagine there are not that many (directly) comparable papers already published, but ultimately the approach presented here has to compete with ALL other succinic production methods in terms of productivity or other performance parameters.

Thank you for your suggestion to include a table comparing the results obtained in this study with previously published works on succinic acid production. We appreciate your feedback and have thoroughly discussed the matter.

We acknowledge the importance of benchmarking our approach against other methods in terms of productivity and other performance parameters. For these reasons, we updated the discussions with other findings to compare our results deeply.

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Nevertheless, after careful consideration, we believe that creating a table to compare all methods of succinic acid production would be better suited for a review or meta-analysis rather than for this study. The reason for this is the significant number of variables involved in different fermentation tests, which makes it challenging to compare all the results.

3)The use of alternative carbon sources is seen as the most promising approach in the introduction, so the question arises as to what solid fraction (section 2.5) remains after solid-liquid separation or what could be done with it? Or to put the question another way - would this residual solid matter interfere with the later fermentation? With regard to your discussion (lines 379-384), it would be interesting to know with what yield the pre-treatment and hydolysis took place or whether >20% of the biomass could also be used.

Thank you for your insightful feedback on our study. We appreciate your interest in the yield of the A. donax hydrolysis treatment. However, it is important to point out that our research focuses on a different aspect. Nevertheless, we understand the significance of this parameter and would like to address your comment.

To strengthen the validity of our findings, we have included citations from previous works to give additional information about the nature and the process for A. donax conversion {#31, #41}. Regarding the addition of 20% of A. donax, it was established as a good amount that could be used in our experimental setup, thanks to previous researchs. We acknowledge your suggestion to explore further percentages of addition with an adapted 4D strain, and we agree that it could be interesting for future investigations. By doing so, we can gain deeper insights into optimizing the process and potentially increasing the overall yield and use of A.donax hydrolisate.

We would like to express our gratitude for your comment, which inspire us on calculating a more comprehensive yield. We kindly indicate you the following lines:399 – 402; where this calculation is discussed so that we provide an overview of the yield of the whole process.

4) The procedure of the different screening and adaptation stages is logical and therefore the following question is not really a criticism, but in view of the relatively small differences between the strains shown in Figure 2, it could be that you would come to different results if you had already carried out the tests (section 2.5) in the bioreactor at constant pH instead of static shake flasks. Could you please comment briefly on this based on your own experience?

Thank you for asking additional information regarding the batch trials. The fermentation process in bioreactors could allow monitoring and control of several parameters such as pH. However, in the specific context of our study, the batch trials were designed to assess the response of adapted bacteria and native strains under progressively increasing stress conditions (view line 183). These stress conditions included increasing concentrations of acids resulting from the fermentation of Arundo donax hydrolysate. Based on our experience, conducting a test in the bioreactor at a constant pH could yield different results and potentially uncover additional differences, as demonstrated in the final experiment comparing the adapted strain with the native one.

5) It is probably just an accidental wording to list SA in the summary (line 19) as a "by-product"...!?

Thank you for pointing out the issue in the abstract regarding the characterization of succinic acid as an end-product of fermentation. We appreciate your keen observation and would like to clarify and rectify this inaccuracy.

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Thank you once again for your time and effort in reviewing our work. We appreciated your valuable input.

Reviewer 3 Report

The study is well organized and includes considerable results. However, due to the following comments, it could be considered for publication after major revision. 

• There are some grammatical errors such as punctuation, long sentences, etc. to be corrected. It is highly recommended to revise the manuscript with an English native editor.  

• Organisms' scientific names should be written in Italic form. Please revise this issue in the whole manuscript. 

• After first appearance, scientific species names should be abbreviated. Please revise this error in the whole manuscript, including the abstract. 

• In microbiology, it is not common to use cell lines for bacteria. It is routine for eukaryotic cell culture. Please replace it with strain or avoid this expression in the whole manuscript (highlighted in blue). 

• As B. succiniciproducens is anaerobic, it is recommended to explain regarding anaerobic culturing method and conditions in subsection 2.1 or 2.2. 

• It is not too common to use log CFU/mL instead of CFU/mL. It is better to convert it to CFU/mL. 

• Could the authors explain why they did not use experimental design software for growth tolerance tests to different inhibitory mixtures? 

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Comments for author File: Comments.pdf

There are some grammatical errors such as punctuation, long sentences, etc. to be corrected. It is highly recommended to revise the manuscript with an English native editor.

Author Response

1) There are some grammatical errors such as punctuation, long sentences, etc. to be corrected. It is highly recommended to revise the manuscript with an English native editor.

We acknowledge and appreciate your attention to detail regarding the grammatical errors, punctuation, and sentence structure. We agree that it is important to ensure the clarity and coherence of the manuscript. To address these concerns, we revised the manuscript.

2) Organisms' scientific names should be written in Italic form. Please revise this issue in the whole manuscript.

We appreciated this kind of thoughtfulness. We fix the italic form for all Organisms'scientific names.

3) After first appearance, scientific species names should be abbreviated. Please revise this error in the whole manuscript, including the abstract.

Thank you very much for your suggestion, we fixed the abbreviations throughout the text wherever possible.

4) In microbiology, it is not common to use cell lines for bacteria. It is routine for eukaryotic cell culture. Please replace it with strain or avoid this expression in the whole manuscript (highlighted in blue).

Thank you for bringing up the terminology concern regarding the use of "cell lines" in the manuscript. We appreciate your insight and agree that the term "cell line" is more commonly associated with eukaryotic cell culture rather than bacteria.

To address this issue, we would like to explain our reasoning behind the use of the term "cell lines". We chose to define the strains undergoing sequential adaptation as "cell lines" to highlight their specific characteristics and emphasize that they are the result of the adaptation process. However, we understand that this terminology may cause confusion and could be misleading.

Taking your suggestion into consideration, we propose replacing the term "cell lines" with "adapted bacterial lines" throughout the manuscript. This modification will help maintain a clear distinction and align with established conventions. Additionally, we will use the term "strain" for BPP7 and BPP8, which were isolated in a previous work, as well as for the strain 4D resulted in this study.

Making these adjustments will ensure that the terminology used in the manuscript accurately reflects the microbiology field's conventions and avoids potential confusion among readers.

5) As B. succiniciproducens is anaerobic, it is recommended to explain regarding anaerobic culturing method and conditions in subsection 2.1 or 2.2.

We included this information in the indicated sections as a fulfilment and improvement of the M&M paragraphs.

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6) It is not too common to use log CFU/mL instead of CFU/mL. It is better to convert it to CFU/mL.

All results were modified and updated according to the requested conversion.

7) Could the authors explain why they did not use experimental design software for growth tolerance tests to different inhibitory mixtures?

Thank you for this question. The decision to not use experimental design software for growth tolerance tests to different inhibitory mixtures was based on several considerations specific of our study.

Firstly, our experimental setup stated with the assumption of adapting Basfia succiniciproducens strains to the individual inhibitors resulting from the hydrolysis of Arundo donax. This initial step allowed us to establish a baseline for further investigation. Subsequently, the adaptation steps were carried out in mixtures containing all inhibitors. This approach allowed us to simulate a more realistic scenario, as the strains would likely encounter a mixture of inhibitors during fermentation trials.

Furthermore, our experimental design aimed to evaluate the response of the various bacterial lines that had been adapted to the inhibitors. Each adaptation target could only proceed if the previous step gave positive results, creating a chain reaction. This sequential approach was essential for systematically assessing the strains' ability to tolerate inhibitory mixtures.

Considering the high variability observed in the strains' responses throughout the adaptation process, we made the decision to rely on direct observations and manual decision-making rather than employing software for experimental designs. Our emphasis was on carefully analyzing and interpreting the experimental outcomes, as this variability demanded a case-by-case evaluation.

In conclusion, due to the specific objectives and constraints of our study, and the importance of informed decision-making based on detailed observations, we chose not to employ experimental design software for growth tolerance tests to different inhibitory mixtures.

We appreciate your question and hope that this explanation clarifies our decisions.

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Thank you once again for your time and effort in reviewing our work. We appreciated your valuable input.

Round 2

Reviewer 2 Report

Thank you very much for the answers and additions to the manuscript. However, the literature in the mentioned period is still too high (>30% "2015 or older") and I would like to motivate you to further improve the citations. Due to the large number of recently published papers, it should not be that difficult to update them further. As an example (and there may be more), you can delete/replace [35], which has nothing to do with succinate production. Apart from that, the improved quality of the revised version is much appreciated.

Author Response

1) Thank you very much for the answers and additions to the manuscript. However, the literature in the mentioned period is still too high (>30% "2015 or older") and I would like to motivate you to further improve the citations. Due to the large number of recently published papers, it should not be that difficult to update them further. As an example (and there may be more), you can delete/replace [35], which has nothing to do with succinate production. Apart from that, the improved quality of the revised version is much appreciated.

Dear,

We would like to express our sincere gratitude for your assistance and all the suggestions.

We are pleased to inform you that after conducting a thorough revision of the bibliography, we have successfully managed to reduce its percentage to below 30% (and within the range of 20 to 25%).

In particular

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The reference #35 was replaced by the reference #37, and other references such as #5-6-7-27 were added / substituted throughout the manuscript.

Once again, thank you for your continuous support and guidance throughout this process.

Reviewer 3 Report

Thanks for your response.

Author Response

Thank you very much for taking the time to re-read our article.