Production of L (+) Lactic Acid by Lactobacillus casei Ke11: Fed Batch Fermentation Strategies
, Daiane Cristina Sass 1 and Jonas Contiero 1,2,*Round 1
Reviewer 1 Report
The topic of the study fits within the scope of the journal, and the experimental work is organized with general methods. But there are still few remaining questions/comments listed below in detail:
Some detailed aspects/questions:
First of all (and this has impact on the sections “introduction/state of the art” and “results and discussion”!): The percentage of the cited literature in the range of “2013 or older” is too high (70%). From that perspective it is recommended to update the references to justify the novelty of the present work more thoroughly. In addition it would be helpful to insert a table of already published figures comparing the here achieved results.
Another general remark concerns the tables 3-5 since the results are illustrated with the figures 2/3. It might be an option to prepare some of the information regarding the experimental details in a supplementary file?!
Because of the importance to provide a high optical purity for subsequent polymerization the question raises if there are any figures available for the here presented investigations? Apropos purity: besides the optical also the chemical purity plays an important role in this context. What about all the other impurities (ashes/ions) during your downstream processing procedure?
As stated in lines 612 the validation of the optimized medium has been carried out in a shaker. Why didn’t you perform these kind of “final experiments” in a fully equipped bioreactor as well? What do you mean by "calcium carbonate to control pH" - is it just a pH adjustment or really some kind of (continuous) control?
Some formalities:
I’m not sure if there is a new requirement by the journal, but actually the order of the sections should be changed… methods should be placed before the results.
It would be useful to harmonize the y-axes of the figures 7&8 for better comparison.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Dear authors,
The paper includes wide information as regards the production of lactic acid and its purification. However, the way the results are presented in most cases is not clear and, at some point, very difficult to follow. You can find below some comments as regards your work:
In general, the introduction section should be revised as information provided is very brief in some cases.
Line 41. Replace obtainment
36-38. Explain more the chemical synthesis with lactonitrile and the viability of the process
Line 41. Explain how it is posible to obtain the D or L enantiomer
Line 55. Explain more about the components that should be present in the media
56-58. Parameters afecting LAB fermentation are exposed, however, a range of values for these parameters should be provided.
Line 60. “this organic acid” should be replaced by “lactic acid”
62-64. Information included in very brief.
Line 68. In the introduction section “pulpe, constant and exponential feeding” should be explained.
Material and method section should appear before results and discussion
Table 1: At some point should be explained the role of Tween 80, citrate, acetate, etc in the medium
Explain the * in sucrose
Figura 1: I am not sure if the data of this figure were obtain in this work as CSL has not been identified before.
Line 87-90. It negative effects of this component have been studied before, why was added to the medium?ç
Line 101. Explain why in experiment 14 the residual reducing sugars is very high. Why those sugars were not fermented to lactic acid?
Line 117. Separate “usedthe”
Line 151. Explain “low density of the inoculum”
Line 165-166. With this concentration of inoculum, which concentration of lactic acid were obtained?
Line 168. In these conditions, which concentration of lactic acid will be produced?
More information should be provided as regards experiments which results are exposed in Table 1 and 2.
Line 172-174. More information should be provided as regards the conditions of this fermentation (temperature, speed, etc).
Figure 4. Experiments should be carried out in duplicate
Figure 4. Why in this experiment, final reducing sugar is zero?
Line 193. You mentioned “fermentation kinetics” but only productivity was calculated.
Line 194-196. You should clarified if CaCO3 was used to control the pH.
Line 213-215. Explain this effect
Figure 6. The line of biomass does not appear in the figure
Line 223-225. Information is not clear
Line 236. I would say lactic acid production, not recovery
Line 249. Explain “diverse effects”
Line 250-256. And what happens for CaCO3?
Line 257-263. There are papers that demostrate that lactic acid is produced at pH 5. Also, you has not mentioned the concentrations of NaOH and CaCO3 used in this paper.
Line 274. Specified the feed used for the experiment not only the feed rate
Line 291. With 44.22 g/L of remaining sugars, the process is not profitable. Why were not these sugars fermented?
It should be mentioned how specific velocities of production are calculated
Line 322. Explain “continual cultivation “
Line 325. What did you mean with “with all culture conditions”?
Line 322-333. More information about this work should be provided
Line 335-338. It should be explaind how was feed the reactor, which concentration of CaCO3 was used.
Line 350. Which concentration was used to form crystals?
Line 351. Replace lactic acid production by lactic acid concentration
Line 353. In that work, LAB were grown at 50 °C?. Explain such a high temperatura
Line 351-354. Explain differences between using 900 or 100 g/L feed solution
Line 355. When you said “These results” you are refering to the ones obtained in your paper. If this is the case this information should appear before the results of Coelho.
Line 362-363. Give more information
Line 634-365. Explain more about findings of Coelho.
Line 373-375. How do you explain these differences.
Table 6 should be modified. The table caption said “highest values”, however, when a, b, c, etc are explained, in some cases it is writen highest values and, in others, not. Also, for letters c and d, it should be explained differences between pulse and constant.
Line 397. Units for 3.74 should be given
Line 398-401. This information is not clear
Line 431. Explain the procedure follows by Bernardo
Line 446-448. Which experiments do you reffert to with “both”?
Line 446-448. Why did you use this broth for the purification?
Table 7. What do you refert to when you said “Final solution”?
Line 501-506. This information should have been shown before. This is not clear
Equation 1: Change to X0
Line 560. At which temperature?
Line 562. Fed batch instead of batch fed
Line 566. Concentration of CaCO3
Line 567. Explain the pulse feeding
Line 568. Which was the feed rate for the constant feeding?
Line 575. CaCO3 subscript
Equation 4. Explain the parameters of the equation
Line 596. Give more information about the method to determine lactic acid (equipment, column, solvents, etc). Moreover, explain how you distinguish between L and L lactic acid.
Modify the conclusions of the paper.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript is very interesting, well-organized and comprehensively demonstrates the performed studies on the production of L (+)- lactic acid by Lactobacillus casei Ke11. I highly recommend this manuscript for publication in its current form.
Author Response
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Round 2
Reviewer 1 Report
The proportion of "old literature" is still too high (>50%)... before I go into the other details, I would like to encourage the authors once again to update the references significantly!
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Dear authors,
I have seen that you have made some changes in the paper considering my comments, however, many comments have not been considered to improve the quality of the paper.
Author Response
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