Well Knowledge of the Physiology of Actinobacillus succinogenes to Improve Succinic Acid Production
Round 1
Reviewer 1 Report
This manuscript describes the knowledge on physiology of A. succinogenes to improve succinic acid production.
In Abstract, the succinic acid yields and concentrations obtained were significant and in the order of those reported in the literature. It is better compare these results with the previous results reported by other researchers in Table.
If the addition of MgCO3 and fumaric acid cause the improvement of SA yields and concentrations, some discussion is needed in a gene level for better understanding.
In Section 3.7.2, Scale-up study, 250 mL to 1L, further to 3 L of bioreactor study could not be the scale-up study, but could be a little bigger. It is better change the word scale-up to “Study in different sizes of bioreactor” etc.
Author Response
Response to Reviewer 1 Comments
Dear Editor,
Thank you for your mail and for the expert Reviewers ‘comments. Please find enclosed the revised manuscript. The points raised by the Referees are clarified below and the manuscript has been revised accordingly.
Point 1: In Abstract, the succinic acid yields and concentrations obtained were significant and in the order of those reported in the literature. It is better compare these results with the previous results reported by other researchers in Table.
Response 1: We totally agree with the Reviewer. A comparison with the literature is added in a new table (table 8). We hope that the modification made at the end of the paper will meet your expectations.
Point 2: If the addition of MgCO3 and fumaric acid cause the improvement of SA yields and concentrations, some discussion is needed in a gene level for better understanding.
Response 2: We totally agree with the comment of reviewer, however, we have no more details regarding what happened on a gene level. In our opinion, more details are not really needed in this paper, since more information can be found in a previous study (Pateraki et al., 2016).
Point 3: In Section 3.7.2, Scale-up study, 250 mL to 1L, further to 3 L of bioreactor study could not be the scale-up study, but could be a little bigger. It is better change the word scale-up to “Study in different sizes of bioreactor” etc.
Response 3: Thank you for this proposal and the correction was done in the revised manuscript. The word is changed from scale-up into study in different sizes of bioreactor.
We hope that the new version of our paper meets the expectations of the Editorial Board of Applied Microbiology Journal. Please do not hesitate to contact us at your convenience if you need further information.
We are looking forward to hearing from you,
Yours sincerely
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In this paper, the authors evaluate the production of Succinate by Actinobacillus succinogenes under different conditions. They test the effect of the carbon source and the addition of MgCo3 to the culture media, as well as Fumaric acid, a precursor of succinic acid.
Although the results are interesting, this manuscript needs to be strongly improved before it is suitable for publication. The introduction is too long, as well as the material and methods section. These latter are too detailed, including formulas for the calculation of widely used parameters, e.g. yield and productivity, the revival of the strain received from DSMZ collection, among other aspects. Some of the methodologies are not clear, like the one presented in lines 164-167, for example. In the results and discussion section, sometimes it is not clear if some statements are based on experimental work presented in this manuscript or are based on previous work described in the literature. This is only perceivable because there is a reference at the end of the sentence (ex. Lines 206-208). The quality of the figures should also be improved, e.g. Figure 9, the graphics are too big, some of the legends on the right axis are missing (ex. Figure 1a and 1 b). Overall this section should be shortened and more objective. I believe that this manuscript would benefit from two distinct sections instead of a combined Result and Discussion. Together, all of these aspects make the manuscript difficult to follow and thus it should be revised and restructured before these data can be published.
In addition, the manuscript should be revised by a proofreader with good written English skills.
Author Response
Response to Reviewer 2 Comments
Dear Editor,
Thank you for your mail and for the expert Reviewers ‘comments. Please find enclosed the revised manuscript. The points raised by the Referees are clarified below and the manuscript has been revised accordingly.
Point 1: In this paper, the authors evaluate the production of Succinate by Actinobacillus succinogenes under different conditions. They test the effect of the carbon source and the addition of MgCo3 to the culture media, as well as Fumaric acid, a precursor of succinic acid.
Although the results are interesting, this manuscript needs to be strongly improved before it is suitable for publication. The introduction is too long, as well as the material and methods section. These latter are too detailed, including formulas for the calculation of widely used parameters, e.g. yield and productivity, the revival of the strain received from DSMZ collection, among other aspects. Some of the methodologies are not clear, like the one presented in lines 164-167, for example. In the results and discussion section, sometimes it is not clear if some statements are based on experimental work presented in this manuscript or are based on previous work described in the literature. This is only perceivable because there is a reference at the end of the sentence (ex. Lines 206-208). The quality of the figures should also be improved, e.g. Figure 9, the graphics are too big, some of the legends on the right axis are missing (ex. Figure 1a and 1 b). Overall this section should be shortened and more objective. I believe that this manuscript would benefit from two distinct sections instead of a combined Result and Discussion. Together, all of these aspects make the manuscript difficult to follow and thus it should be revised and restructured before these data can be published.
In addition, the manuscript should be revised by a proofreader with good written English skills.
Response 1: Thank you for your comment, we have tried to reduce and improve the chapter’’ Introduction’’ and “Materials and Methods”, following the recommendations of the Reviewer. However, for a better comprehension we prefer to give the corresponding information (formulas for the calculation) in the full form. For the methodology presented in lines (137-139), it corresponds to a simple titration method which is a technique used in chemistry to help determine the concentration of a reactant mixed within an unknown solution. The process involves adding a known solution to the unknown solution until a reaction occurs. Most often, this reaction is a color change. In the results and discussion section, our results are first provided ‘’ Fermentations of A. succinogenes in micro-aerobic conditions did not show any SA production, while it revealed low yields of (< 0.25 mol-C SA/mol- CGLU) and productivities (< 0.012 g-SA/L/h), when 0.04 mol L-1 of glucose was used as the only carbon source in anaerobic conditions, SA was produced at low levels, recording a value of 0.01 mol L-1 (Fig 1-a)’’. After that our results are agreed with what mentioned in previous work ‘’ Further evidence is provided in previous study [31, 32] demonstrating that SA is formed even when this substrate was added as the sole carbon feedstock in anaerobic conditions.’’ The quality of all figures are improved. Indeed, the manuscript would benefit from two distinct sections instead of a combined Result and Discussion. However, to avoid overloading this paper, which is rather quite long, the chapter results and discussion are combined in on section study. We hope that the new version of our paper meets the expectations of the Editorial Board of Applied Microbiology Journal.
Please do not hesitate to contact us at your convenience if you need further information.
We are looking forward to hearing from you,
Yours sincerely,
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript represents an exploratory research about optimizing SA production by A. succinogenes. While the discussion into metabolic pathways is deep, and topic could be interesting to a broad audience, the presentation quality in the result section (long paragraphs, over complicated sentences..) and the lack of information in figures & tables (experimental details in legend, biomass data...) could be improved. Here are some specific comments:
Line 88-91: The sentence is too long to follow: do authors mean that SA production is improved by the combination of substrate use, carbon partitioning and optimization of carbon flux? Or each approach is tested individually?
Line 129-131: "A series of experiments... fumaric acid (FA) as co-factor in various concentrations", what were the concentrations of FA added? what were the final concentrations in the fermentation media?
Line 178: How the verification by NMR was performed? Please briefly describe instrument used, acquisition mode and quantification procedures.
Line 191-192: dS/dt, dP/dt is misleading. The formula showed average substrate consumption/production rate over the course of fermentation, not exactly real-time rate as derivative usually means.
Line 221: Authors explained well the SA/Glucose yield increase, however, no interpretation is given in the paragraph for the biomass yield increase by MgCO3
Paragraph 3.2: While the title of the paragraph is about mixed sugar conditions, authors presents fructose data only in Figure 2, and Table 1 attempts to compare glucose-only media A against fructose media B. No interpretation is given about the difference between the two sugars.
Line 273-274: Authors claim that "combining two sugars consume more quickly" - the consumption is not show in Figure 3. They should compare Figure 3 clearly the sugar consumption, biomass yield and succinate/sugar yield between mixed sugar and mono sugar conditions. Now the paragraph and interpretation is highly confusing.
Line 335: "Fumarate uptake of 100mg/L...substrate specificity for fumarate but not for succinate". What do authors mean here?
Table 3: What are the runs 1, 2 and 3? Media A+B was used in run 1? Such details should be explained in the table legend. The biomass yield is not presented in the table. Do fumarate influence cell growth?
Line 349-350: the sentence is not clear. Still not clear what "extra SA" means. In addition, throughout the paragraph, "error percentage" is used. Does authors mean experimental variation or the deviation to theoretical values (which error percentage normally means) or difference to control experiments?
Table 4: Please repeat in the legend what run 1,2..5 represents. Please compare biomass data between runs.
Section 3.5: Authors made several hypotheses about metabolism of SA production, attempting to find the correct hypothesis by comparing the theoretical estimation via MBA and experimentally measured conversion rate. They use long paragraphs and sentences combining pathways, equations and calculations therefore very hard to follow. Could authors provide a table comparing experimental vs estimated conversion rate under different hypothesis?
Table 5: Could the difference in fermentation time be explained?
Line 487-490: The pentose phosphate pathway seems to be major mechanism of SA formation, yet it is not highlighted in Figure 4a.
Section 3.6: Why was the fumaric acid not added as co-factor in 1L experiments?
Figure 7: Comparing with figure 6, a similar concentration of SA (0.4 mol/L) was produced with/without fumaric acid addition. This value is higher than 250mL experiment. What's the reason behind?
Table 7: In 1L bioreactor, the SA obtained was only 0.28 mol/L, while in Figure 6, the final SA was around 0.4 mol/L. What is the difference between two experiments? Was fumaric acid added in this experiment?
Author Response
Response to Reviewer 3 Comments
The manuscript represents an exploratory research about optimizing SA production by A. succinogenes. While the discussion into metabolic pathways is deep, and topic could be interesting to a broad audience, the presentation quality in the result section (long paragraphs, over complicated sentences..) and the lack of information in figures & tables (experimental details in legend, biomass data...) could be improved. Here are some specific comments:
Point 1: Line 88-91: The sentence is too long to follow: do authors mean that SA production is improved by the combination of substrate use, carbon partitioning and optimization of carbon flux? Or each approach is tested individually?
Response 1: We totally agree with the Reviewer and we have tried to improve the sentence, following the recommendations of the Reviewer. Each approach was tested individually, then they are combined.
Point 2: Line 129-131: "A series of experiments... fumaric acid (FA) as co-factor in various concentrations", what were the concentrations of FA added? what were the final concentrations in the fermentation media?
Response 2: Thank you for your comment. The different concentration of fumaric acid is presented in the table and they are added in the revised manuscript, their final concentrations are not calculated however, the % of FA consumption are provided in the tables.
Point 3: Line 178: How the verification by NMR was performed? Please briefly describe instrument used, acquisition mode and quantification procedures.
Response 3: NMR (Nuclear Magnetic Resonance) was used to prove and to confirm the production of succinic acid by Actinobacillus. The spectra are recorded on a Bruker Avance III 400 spectrophotometer operating at 400.13 MHz for 1H, equipped with BBFO probe with a Z-gradient coil and a GREAT 1/10 gradient unit. The standard temperature was adjusted to 298 K. All NMR tubes were made as follows:
- Collection of 450 μl of solution and introduction into NMR tube.
- Addition of 100 μl of D2O to NMR (to remove water)
-Homogenization in NMR tube.
-Spectrum recording: Three experiments per test tube were performed in a systematic way, with the autosampler:
1) Recording a 1H spectrum in 80 scans
2) Recording of a 1H spectrum with presaturation of water in 80 scans
3) Fast HSQC to check the 13C allocation of the proton peak at 2.4 ppm
The NMR spectra were manually phased and baseline corrected using MestReNova (Mnova). Each NMR spectrum was used to construct a data matrix by subdividing it into regions having an equal bin size of 0.5 ppm over a chemical shift range of 1-9 ppm.
Point 4: Line 191-192: dS/dt, dP/dt is misleading. The formula showed average substrate consumption/production rate over the course of fermentation, not exactly real-time rate as derivative usually means.
Response 4: Thank you for your comment, exactly, dS/dt, dP/dt are the average substrate consumption/production rate over the fermentation time and not the specific rate of consumption/production.
Point 5: Line 221: Authors explained well the SA/Glucose yield increase, however, no interpretation is given in the paragraph for the biomass yield increase by MgCO3
Response 5: Thank you for your comment, we have tried to improve paragraph following the recommendations of the Reviewer as follows ‘’The addition of MgCO3 was beneficial to promote the biomass yield and succinic acid synthesis. This could be attributed to the necessity of the CO2 and the cofactor Mg2+ provided by MgCO3 for conversion pathway into biomass and products’’.
Point 6: Paragraph 3.2: While the title of the paragraph is about mixed sugar conditions, authors presents fructose data only in Figure 2, and Table 1 attempts to compare glucose-only media A against fructose media B. No interpretation is given about the difference between the two sugars.
Response 6: We totally agree with the Reviewer and the correction was done in the title of the paragraph as follows ‘’Results from 250 mL anaerobic bottles using sole and mixed sugars as carbon source in anaerobic conditions’’. The aim of this paragraph was to give information about the possible use of glucose or fructose as sole carbon source by A. succinogenes at different concentrations, then to combine this two sugars at different concentration to prove that our bacteria are able to consume different source of sugars at different concentrations.
Point 7: Line 273-274: Authors claim that "combining two sugars consume more quickly" - the consumption is not show in Figure 3. They should compare Figure 3 clearly the sugar consumption, biomass yield and succinate/sugar yield between mixed sugar and mono sugar conditions. Now the paragraph and interpretation is highly confusing.
Response 7: A figure was added (figure 3a) to the revised manuscript comparing the sugar consumption, succinic acid concentration and the succinic acid yield.
Point 8: Line 335: "Fumarate uptake of 100mg/L...substrate specificity for fumarate but not for succinate". What do authors mean here?
Response 8: Using UNIPROT database, A. succinogenes have a transport system of 3 transporters for fumarate uptake and expressed during anaerobic growth on fumarate; they are: Asuc_1063, Asuc_1999, and Asuc_0142. So, A. succinogenes grows well on fumarate under anaerobic conditions. This was confirmed experimentally by supplying different concentrations of fumarate. Supplied fumarate is almost turned into succinate. However, the bacteria could not uptake the succinate or consume it as carbon source. For this reason, the systems had substrate specificity for fumarate even at low concentration of 0.0008 mol. L-1 (= 100 mg/L) of fumarate and not for succinate.
Point 9: Table 3: What are the runs 1, 2 and 3? Media A+B was used in run 1? Such details should be explained in the table legend. The biomass yield is not presented in the table. Do fumarate influence cell growth?
Response 9: All these corrections were made in the revised manuscript. The biomass yield was not calculated here because our aim was to see the effect of the fumaric acid on the succinic acid production. According to the OD measured at 660 nm in the table 2, we can see clearly that fumarate influence the cell growth (when the concentration of FA increase, the OD decrease, this could be attributed to the fact of addition of acid (fumaric acid) into the media).
Point 10: Line 349-350: the sentence is not clear. Still not clear what "extra SA" means. In addition, throughout the paragraph, "error percentage" is used. Does authors mean experimental variation or the deviation to theoretical values (which error percentage normally means) or difference to control experiments?
Response 10: The correction was made. The extra SA means the succinic acid that came from fumaric acid (excluded from the succinic acid produced from glucose and fructose). So first, test was run without fumaric acid to see the succinic acid concentrations produced, then in the same condition and parameters, fermentation test was run in the addition of fumaric acid (so the succinic acid concentration coming from glucose and fructose are known and the rest which is called extra SA is coming from fumarate). The percentage of difference is calculated as follows: % of Difference= ((FA consumed – SA produced)/FA consumed) *100
Point 11: Table 4: Please repeat in the legend what run 1,2..5 represents. Please compare biomass data between runs.
Response 11: All these corrections were made in the revised manuscript. Biomass data was not calculated for all the runs in the table 4, for this reason we did not perform a comparison.
Point 12: Section 3.5: Authors made several hypotheses about metabolism of SA production, attempting to find the correct hypothesis by comparing the theoretical estimation via MBA and experimentally measured conversion rate. They use long paragraphs and sentences combining pathways, equations and calculations therefore very hard to follow. Could authors provide a table comparing experimental vs estimated conversion rate under different hypothesis?
Response 12: Thank you for your proposal. However, in our opinion, it was not possible to combine all these pathways and their equations and calculations to compare experimental vs estimated conversion rate especially when more than pathway are active.
Point 13: Table 5: Could the difference in fermentation time be explained?
Response 13: Table 5 present the different runs mentioned in the section ‘’results and discussion’’, each experiments were tested during a defined time. Since the first part was only a preliminary part of this study: the objective was only to get information about the production of SA and not to study the effect of fermentation time. However, in our opinion, fermentation time between the results would not affect the analysis of the results.
Point 14: Line 487-490: The pentose phosphate pathway seems to be major mechanism of SA formation, yet it is not highlighted in Figure 4a.
Response 14: Fig 4a present the carbon flux or the metabolic pathway in anaerobic conditions while the pentose phosphate pathway is occurred in the oxidative branch and not in the reductive branch of Tricarboxylic cycle acid. For this reason, it is not highlighted in this figure.
Point 15: Section 3.6: Why was the fumaric acid not added as co-factor in 1L experiments?
Response 15: Thank you for your comment, fumaric acid was not added first in the 1L experiments because that the aim of this experiments to more deeply investigate SA production, by performing batch cultures under anaerobic conditions in 1 L reactor employing SM containing glucose and fructose and or mixtures of both sugars to have different initial concentration varied in total between 0.1 and 0.4 mol L-1.
Point 16: Figure 7: Comparing with figure 6, a similar concentration of SA (0.4 mol/L) was produced with/without fumaric acid addition. This value is higher than 250mL experiment. What's the reason behind?
Response 16: Figure 6 was removed to avoid confusion, the results obtained in figure 7 were similar to those obtained in 250 mL anaerobic bottles.
Point 17: Table 7: In 1L bioreactor, the SA obtained was only 0.28 mol/L, while in Figure 6, the final SA was around 0.4 mol/L. What is the difference between two experiments? Was fumaric acid added in this experiment?
Response 17: The correction was made, (fig 6 was removed to avoid confusion).
A careful reading of the manuscript has been performed and several corrections can be found throughout the manuscript. We hope that the modification made will meet the expectations of the Reviewer.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
This manuscript was properly revised and now it is acceptable.
Reviewer 3 Report
The quality of presentation is improved.
I recommend publication of current version.
