Use of Corn-Steep Water Effluent as a Promising Substrate for Lactic Acid Production by Enterococcus faecium Strain WH51-1
, Salem S. Salem 1, Amr Fouda 1, Mamdouh S. El-Gamal 1 and Mohamed Ali Abdel-Rahman 1,2,*Round 1
Reviewer 1 Report
Comments:
Overall:
-Extensive English and formatting editing required. Minor consistent things such as some references are bolded while others are not, or inconsistent patterns when beginning a sentence with a citation (eg; ‘Watson [40] (line 204)’ compared to ‘[41] and [39] reported…(line 209)’) show a lack of editing done by the authors prior to submission. As well, the same acronyms are introduced multiple times throughout the manuscript. References are also out of date – with line 37 showing market analysis from 2016-2025, and no mention of the current market at the midpoint of this range for the current date. This would have shown if the estimates in that reference were accurate and deserving to be used, or if others should have been sought.
-This manuscript dedicates a significant portion of the text to the analysis of the CSW. However, this is not novel, and as CSW is a waste substrate there would be variability between production locations, or even between batches at the same production facility. As such, this type of analysis should be considered routine work before beginning any study with a waste substrate.
-This is not an optimization. There is no study of the interaction of the four factors investigated, or any statistical analysis performed. As a result, this would be considered the initial first steps of optimization, which should be followed by more robust methods as outlined in reference 18 for example.
-Overall, this new isolate is indeed interesting, however this does not meet the merit of publication. Some of the results are not statistically different from one another (for example, NaOH vs CaCO3 CFU/mL are within each others standard deviations, not an increase as claimed on line 424). Others are difficult to understand the significance – for example, the inoculum size. Studying the total viable cells at the same timepoint regardless of substrate availability or microbial activity is inaccurate at best. Why not choose a more robust and reproduceable indicator such as depletion of substrate availability? In addition, no information regarding the preculturing methods for the batch fermentations was given.
-For these outlined reasons alone, this manuscript must be rejected and extensively revised with significant work done in the lab before resubmission.
Author Response
Response to Reviewers Report-1
(fermentation-1262240)
To Reviewer #1
Thank you very much for reviewing our manuscript and valuable comments. We are also grateful for the favorable comments. We made corrections and we hope they meet with your approval. A detailed explanation is given below:
Reviewer comment #: Extensive English and formatting editing required.
Author response #: Thank you for your comment. We have completely revised the manuscript and corrected several errors. We hope that the revised version meets your approval and agreement.
Reviewer comment #: Minor consistent things such as some references are bolded while others are not, or inconsistent patterns when beginning a sentence with a citation (eg; ‘Watson [40] (line 204)’ compared to ‘[41] and [39] reported…(line 209)’) show a lack of editing done by the authors prior to submission. As well, the same acronyms are introduced multiple times throughout the manuscript.
Author response #: We are very sorry for this mistake as we did not notice this problem in the submitted Manuscript after final correction by the “Endnote” program for reference style. In the revised version, we have carefully checked the names of authors before the number of references.
Reviewer comment #: References are also out of date – with line 37 showing market analysis from 2016-2025, and no mention of the current market at the midpoint of this range for the current date. This would have shown if the estimates in that reference were accurate and deserving to be used, or if others should have been sought.
Author response #: Thank you very much for your valuable comment. We have corrected and updated this information as you indicated. We added the following sentence “The global market size for LA was valued at USD 2.7 billion in the year 2020 and is expected to expand at a compound annual growth rate (CAGR) of 8.0% from 2021 to 2028 (https://www.grandviewresearch.com/).
Reviewer comment #: This manuscript dedicates a significant portion of the text to the analysis of the CSW. However, this is not novel, and as CSW is a waste substrate there would be variability between production locations, or even between batches at the same production facility. As such, this type of analysis should be considered routine work before beginning any study with a waste substrate.
Author response #: Thank you for your comment. The analysis of the CSW is not novel and we did not say in the manuscript, but the novelty of this manuscript in there is no study demonstrating the use of CSW as the main carbon source for LA production till this time, due to not only it is containing high sugar and nutrients content but also, the existence of high content of inhibitory compounds for microbial growth in the concentrated raw material. In previous studies, CSW has been used only as nitrogen source supplementation for LA at low concentrations. But our study is the first to use the whole effluent as a carbon and nitrogen source. Also, the novelty is that our selected strain is highly resistant to the main inhibitory compounds existed in the CSW and could efficiently utilize it to a high titer of LA.
As well, according to your comment “CSW is a waste substrate there would be variability between production locations, or even between batches at the same production facility” were collected several samples from the outlet of a steeping tank at different batches then were mixed carefully then stored at ‒20 °C for further analysis.
Reviewer comment #: This is not an optimization. There is no study of the interaction of the four factors investigated, or any statistical analysis performed. As a result, this would be considered the initial first steps of optimization, which should be followed by more robust methods as outlined in reference 18 for example.
Author response #: Thank you for your comment. This would be considered the initial first steps of optimization as (one factor at a time optimization protocol), which should be followed by more robust methods. We are planning to do statistical optimization experiments to maximize the sugar utilization and LA production from CSW as raw material. But in the current study, we mainly focused on studying the feasibly of the CSW effluent as a cheap alternative substrate for biorefinery to lactic acid. We also aimed to find a potential strain capable to grow and convert sugars in CSW to LA-based on a specific screening protocol. We have successfully obtained this potential strain as WH51-1 and well-characterized and identified it for future studies that will be planned soon. So our planning for this study was not only further optimize the four mentioned factors, but also investigate for the effect of removing the extra supplementation of any nutrient to the CSW and using it as sole media. We also planned to study the effect of various fermentation strategies for the final goal of enhancing the productivity of LA with the complete utilization of sugar content in the waste effluent.
Reviewer comment #: Overall, this new isolate is indeed interesting, however this does not meet the merit of publication. Some of the results are not statistically different from one another (for example, NaOH vs CaCO3 CFU/mL are within each other’s standard deviations, not an increase as claimed on line 424). Others are difficult to understand the significance – for example, the inoculum size. Studying the total viable cells at the same time point regardless of substrate availability or microbial activity is inaccurate at best. Why not choose a more robust and reproduce able indicator such as depletion of substrate availability? In addition, no information regarding the pre culturing methods for the batch fermentations was given.
Author response #: Thank you for your comment, some of the results are not statistically different from one another (for example, the use NaOH and CaCO3), therefore we completely rephrased this section to show the comparable results obtained by both neutralizing agent and give recommendations for NaOH application from the environmental point of view to avoid Gypsum waste formation if CaCO3 is utilized.
As you indicated, we have just studied the total viable count as the only possible way to analyze the actual cell growth in the waste material (CSW). We couldn’t analyze optical density due to the deep browning of the CSW waste and the high TSS content. One more thing, we have analyzed the substrate availability by measuring the total sugar and calculated the residual and consumed sugars as indicated in the Tables. Based on both data of cell growth and substrate availability we could get a clear image of the actual fermentation and kinetic parameters. We could analyze of the actual fermentation parameters and enhancement or reduction of microbial growth. We could conclude the main four optimal factors as the basis for future studies and the actual challenges faced by the efficient utilization of CSW effluent.
At last, we have added the details of seed culture, refreshment, and pre-culture for batch fermentation in the “Material and method” section
Finally, we would like to thank you so much for your valuable recommendations and comments and hope the revised version meets your final approval.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The article is devoted to lactic acid production by a new strain of Еnterococcus faecium in a medium with a new composition, corn steep water. Indeed, ways have recently been sought to obtain valuable metabolites from cheaper substrates. That's why the article makes sense. However, it must undergo significant changes to convince the public of the reliability of the results obtained. Most importantly, "Materials and Methods" should be improved. The composition of "Modified yeast extract dextrose (mYD) medium"may be described in separate paragraph "Fermentation medium". I think that some parts of "Substrate analysis" should be moved to "Аnalytical methods", such as HPLC, Mass Spec, etc. There is no method mentioned for determining fats. HPLC analyzer's model was not mentioned. I think that Figure 1 is not worth showing in the text, it can be shown as an additional file. Minor remarks: the tables in the text should be referred to as Table 1 and not Table (1). Many of the references in the text are bold, but you shouldn't. Table 4 could be given as landscape. At line 107 "gm" should be corrected to "g", lines 178-179, 322, etc.: "hr" to "h". After the first mentioning, "Enterococcus faecium" should be "E. faecium" in the text. Table 3: "Ɣ3Aminobutyric acid" should be changed to Ɣ-Aminobutyric acid". Line 386: please, begin the sentence with the name of [8]. The same, in lines 414, 416, etc. Table 6, first column, please, correct "Incula". The names of Lactobacillus species in the discussion should be according to the new taxonomy from 2020, for instance Lactobacillus casei is now Lacticaseibacillus casei, Lactobacillus paracasei is Lacticaseibacillus paracasei, etc.Author Response
Response to Reviewers Report-2
(fermentation-1262240)
Thank you very much for reviewing our manuscript. We are also grateful for your valuable positive comments.
Reviewer comment #: The article is devoted to lactic acid production by a new strain of Еnterococcus faecium in a medium with a new composition, corn steep water. Indeed, ways have recently been sought to obtain valuable metabolites from cheaper substrates. That's why the article makes sense. However, it must undergo significant changes to convince the public of the reliability of the results obtained.
Author response #: Thank you for your agreement and helpful comments and recommendations.
Reviewer comment #: Most importantly, "Materials and Methods" should be improved.
Author response #: Thank you so much. We have improved "Materials and Methods" and reconstructed and clarified some parts.
Reviewer comment #: The composition of "Modified yeast extract dextrose (mYD) medium"may be described in separate paragraph "Fermentation medium".
Author response #: Thank you for your comment. We have described it in a separate paragraph as you recommended.
Reviewer comment #: I think that some parts of "Substrate analysis" should be moved to "Аnalytical methods", such as HPLC, Mass Spec, etc.
Author response #: As you recommended, we have moved "Substrate analysis" to the "Аnalytical methods" section.
Reviewer comment #: There is no method mentioned for determining fats.
Author response #: Thank you for your comment. There is no method mentioned for determining fats, because we focused on carbon source (as the main substrate for lactic acid production) and nitrogen sources (as a nutritional source for LAB) for the growth of fastidious lactic acid bacteria. So, the main target of lactic acid bio-production is sugars and nutrients (nitrogen, amino acids, trace elements, and vitamins) that can stimulate bacterial growth. Besides this, our Data show the absence of fat-soluble vitamins (Vitamins E, D, A) in corn steep water and this may be due to bad solubility in water, in addition, they compose just a small fraction in CSW as shown in some literature.
Reviewer comment #: HPLC analyzer's model was not mentioned.
Author response #: Thank you so much. We have mentioned the analyzer model as you indicated.
Reviewer comment #: I think that Figure 1 is not worth showing in the text, it can be shown as an additional file.
Author response #: Thank you for your comment. We removed this figure from the manuscript as you indicated.
Reviewer comment #: Minor remarks: the tables in the text should be referred to as Table 1 and not Table (1).
Author response #: We thank the reviewer for your observation. It is corrected and unified throughout the manuscript.
Reviewer comment #: Many of the references in the text are bold, but you shouldn't.
Author response #: We thank reviewer for your observation. All references are corrected as you indicated.
Reviewer comment #: Table 4 could be given as landscape.
Author response #: We thank the reviewer for your recommendation. We changed the Table layout to landscape as you recommended.
Reviewer comment #: At line 107 "gm" should be corrected to "g", lines 178-179, 322, etc.: "hr" to "h".
Author response #: Thank you so much. It is corrected and standardized throughout the manuscript.
Reviewer comment #: After the first mentioning, "Enterococcus faecium" should be "E. faecium" in the text.
Author response #: We thanks the reviewer for your observation. It is corrected and standardized throughout the manuscript.
Reviewer comment #: Table 3: "Ɣ3Aminobutyric acid" should be changed to Ɣ-Aminobutyric acid".
Author response #: We are sorry for this mistake. Ɣ3Aminobutyric acid" is changed to Ɣ-Aminobutyric acid as you indicated
Reviewer comment #: Line 386: please, begin the sentence with the name of [8]. The same, in lines 414, 416, etc.
Author response #: We thanks the reviewer for your observation. We have carefully edited the Manuscript and confirmed the author’s names before the reference number.
Reviewer comment #: Table 6, first column, please, correct "Incula".
Author response #: We thank reviewer for your observation. It is corrected.
Reviewer comment #: The names of Lactobacillus species in the discussion should be according to the new taxonomy from 2020, for instance Lactobacillus casei is now Lacticaseibacillus casei, Lactobacillus paracasei is Lacticaseibacillus paracasei, etc.
Author response #: We thank you so much for your recommendation. We corrected all as you indicated.
Finally, we hope that the revised version meets your approval.
Author Response File: Author Response.pdf
