Improved Raoultella planticola Strains for the Production of 2,3-Butanediol from Glycerol
, Silvia Segarra 2, Alejandro Montesinos 2, Marta Tortajada 2, Daniel Ramón 2 and Antonia Rojas 2,*Round 1
Reviewer 1 Report
The manuscript “Improved Raoultella planticola strains for the production of
2,3-butanediol from glycerol” has focused on enhancing the bioconversion of glycerol to 2,3-BD through applying modifications to the cultivation conditions for R. planticola and also screening the performance of random mutants of the aforementioned microorganism. The screening results presented are interesting and worth publishing, although, the type of evaluation work done lacks pure novelty. The manuscript can be considered for publication in Fermentation after an extensive revision:
Page1-line24-27: These are very basic information that are unnecessary to be presented in the introduction.
Page2-line49: Agricultural waste are different types of biomass. Therefore, they should not be presented separately.
Page3-line98: It has been mentioned that 4 parameters have been considered, glycerol concentration, CoCl2 concentration and temperature. What is the 4th parameter considered?
Please clearly mention in the materials and methods section of the manuscript how many times the flask fermentations and bioreactors have been replicated. Also mention at what certainty level standard deviations have been measured and what error bars present on the column charts.
Page4-line184-186: Although it has been mentioned that the higher temp of 33-37 degrees is optimum for cell growth, it should be clearly justified why temperatures between 28-33 has been examined in this research work?
Page4-line185: add the Reference literature for claims on optimum cell growth and fermentation temperature.
Page5-line188: use single terminology either “glycerin” or “glycerol” throughout the manuscript.
There is a confusion on what is the capacity of the reactor used is and what is the working volume? 1.5 or 1 liter?
Include the line graph of changes in the concentration glycerol vs. time, concentration of 2,3-BD vs. time and pH vs. time.
Page6-line221: what do you mean by “these selected strains were tested again to confirm …” are the rest of the test performed in singles of replicated?
Page7-line241-249: It should be clearly mentioned why the performance in flasks was not as obtained for the richer medium? Mentioned what parameter has contributed to higher yield of BD?
Page7-line251-252: bring references.
Page7-line263: Clearly mention the conclusion on if the presence of acetic acid is favorable as results in higher BD or is a by-product that reduces the yield of BD?
Page8-line 281: Please remove the phrase “robust” as the new mutant organisms are not robust in preserving their performance in long run and are therefore not suitable for industrial applications.
Author Response
The manuscript “Improved Raoultella planticola strains for the production of
2,3-butanediol from glycerol” has focused on enhancing the bioconversion of glycerol to 2,3-BD through applying modifications to the cultivation conditions for R. planticola and also screening the performance of random mutants of the aforementioned microorganism. The screening results presented are interesting and worth publishing, although, the type of evaluation work done lacks pure novelty. The manuscript can be considered for publication in Fermentation after an extensive revision:
Page1-line24-27: These are very basic information that are unnecessary to be presented in the introduction.
Response 1: Yes, thank you for this comment, we have removed the unnecessary information.
Page2-line49: Agricultural waste are different types of biomass. Therefore, they should not be presented separately.
Response 2: The biomass term has been removed
Page3-line98: It has been mentioned that 4 parameters have been considered, glycerol concentration, CoCl2 concentration and temperature. What is the 4th parameter considered?
Response 3: It was a mistake, only 3 parameters were considered, it has been corrected in the text
Please clearly mention in the materials and methods section of the manuscript how many times the flask fermentations and bioreactors have been replicated. Also mention at what certainty level standard deviations have been measured and what error bars present on the column charts.
Response 4: the assays were repeated three times, it has been clarified in the text. The error bars represent standar deviation.
Page4-line184-186: Although it has been mentioned that the higher temp of 33-37 degrees is optimum for cell growth, it should be clearly justified why temperatures between 28-33 has been examined in this research work?
Response 5: Temperatures between 28-33 were chosen because it was found in literature that it resulted in a substantial reduction in ethanol synthesis in favour of 2,3-BD formation. This information and reference have been added to the text.
Page4-line185: add the Reference literature for claims on optimum cell growth and fermentation temperature.
Response 6: Thanks, these references have been added.
Page5-line188: use single terminology either “glycerin” or “glycerol” throughout the manuscript.
Response 7: Thanks, glycerin has been replaced by glycerol.
There is a confusion on what is the capacity of the reactor used is and what is the working volume? 1.5 or 1 liter?
Response 8: The capacity of reactors is 1.5L, and the working volume is 0.5 L.
Include the line graph of changes in the concentration glycerol vs. time, concentration of 2,3-BD vs. time and pH vs. time.
Response 9: we cannot produce this line graph because we compared cultures at 24 hours and do not have exhaustive sampling; it is important to obtain the fastest producers as well. We are preparing a new publication in which exhaustive sampling and comparisons of feeding policy will be shown.
Page6-line221: what do you mean by “these selected strains were tested again to confirm …” are the rest of the test performed in singles of replicated?
Response 10: It means to confirm the results at higher scale, it is now better explained in the text.
Page7-line241-249: It should be clearly mentioned why the performance in flasks was not as obtained for the richer medium? Mentioned what parameter has contributed to higher yield of BD?
Response 11: It has been clarified in the text; the parameter that contributes more to a higher yield is the glycerol concentration, according to the Taguchi results.
Page7-line251-252: bring references.
Response 12: Thank you, they have been included.
Page7-line263: Clearly mention the conclusion on if the presence of acetic acid is favorable as results in higher BD or is a by-product that reduces the yield of BD?
Response 13: It has been clarified now in the text: it is a by-product that reduces the yield to 2,3-BD and should be avoided.
Page8-line 281: Please remove the phrase “robust” as the new mutant organisms are not robust in preserving their performance in long run and are therefore not suitable for industrial applications.
Response 14: Ok, their suitability for long-term industrial application has not been tested in this article, therefore, of course we remove “robust”.
Reviewer 2 Report
I suggest publication of this manuscript in journal : Fermentation after minor revision.
My suggestion:
In analytical method section authors listed HPLC method for determination of by-products (succinic acid, lactic acid, acetic acid, acetoin and ethanol). Table 2 are summarized results obtained in flask and bioreactors cultures. Concentrations of succinic acid, lactic acid, acetic acid in these cultivations are missed. Authors should be added information about concentrations of these products in section Result and Discussion.
Author Response
In analytical method section authors listed HPLC method for determination of by-products (succinic acid, lactic acid, acetic acid, acetoin and ethanol). Table 2 are summarized results obtained in flask and bioreactors cultures. Concentrations of succinic acid, lactic acid, acetic acid in these cultivations are missed. Authors should be added information about concentrations of these products in section Result and Discussion.
RESPONSE 1: Concentration of organic acids such as succinic acid, lactic acid and acetic acid are very low or not detected under the conditions of the assays, particularly in bioreactor tests. Only acetoin and ethanol were detected by HPLC method.
Round 2
Reviewer 1 Report
Considering the revision applied by the authors the manuscript is qualified for publication in Fermentation.
