Enhancement of Growth and Paramylon Production of Euglena gracilis by Upcycling of Spent Tomato Byproduct as an Alternative Medium
Sergio A. Guerrero
Round 1
Reviewer 1 Report
The manuscript may be accepted for publication after a minor revision. Please correct:
-page 2, line 80, the references should be written in the same way (number in squared brackets)
-page 4, equations 2 and 3: the abbreviations PM and CD should be explained or put in the text
-page 7: Table 3 instead of Table 1, Figure 4 instead Figure 3. Please check the numeration of tables and figures in the manuscript. Please put correct title of the table.
-page 7, line 238-242 Should these sentences be in the text?
-page 8, line 274-275 This sentence should have a reference/references.
-page 8, section from 283-302, Please check if this section could be at the beginning of “Discussion”. It`s only suggestion.
-page 9, line 311-318, This section should be in different place.
-The discussion section should be rewritten and put in correct order, because now it is mixed up.
-Q: Why all experiments were performed in duplicates not in triplicates?
Author Response
We, all the authors, appreciate the critical feedback from the editor and the reviewers. We have reviewed the entire manuscript carefully and wrote responses to all issues mentioned. All the mismatched or ambiguous description have revised. The answers have responded to all questions and comments raised by the reviewers. Thank you for your comments and suggestions.
Point 1: The manuscript may be accepted for publication after a minor revision. Please correct:
-page 2, line 80, the references should be written in the same way (number in squared brackets)

Response 1: As commented, we have changed the reference citation type to the number in squared brackets (line 82 in p.2).
Point 2: page 4, equations 2 and 3: the abbreviations PM and CD should be explained or put in the text
Response 2: As commented, we put the abbreviations in the body text (line 148 and 152 in p.4).
Point 3: page 7: Table 3 instead of Table 1, Figure 4 instead Figure 3. Please check the numeration of tables and figures in the manuscript. Please put correct title of the table.
Response 3: As commented, the numbering of the table and figure was corrected (Table 3 and Figure 4).
Point 4: page 7, line 238-242 Should these sentences be in the text?
Response 4: As commented, the sentences were deleted (line 254-256 in p.8).
Point 5: page 8, line 274-275 This sentence should have a reference/references.

Response 5: As commented, the reference citation was inserted into the sentence (line 292 in p.8).
Point 6: page 8, section from 283-302, Please check if this section could be at the beginning of “Discussion”. It`s only suggestion. In page 9, line 311-318, This section should be in different place. The discussion section should be rewritten and put in correct order, because now it is mixed up.
Response 6: For a better understanding of the Discussion section, we put subtitles as follows:
4.1 Impact of carbon source type and concentration
4.2 Application of spent tomato byproduct as an alternative medium
4.3 The impact of initial pH for value-added production from E. gracilis using STB
Point 7: Why all experiments were performed in duplicates not in triplicates?
Response 7: Because of the air flow controller limitation, we had to set two reactors per initial pH condition and substrate as biotic replications, but still we had two sample analyses per each reactor as abiotic replications. Thereby the number of samples analyzed for each single data point was four (n=4).
Author Response File: 
Author Response.docx
Reviewer 2 Report
The research article presents a valorization of tomato waste to produce Euglena gracilis biomass towards the production of paramylon. the topic is novel and the findings are interesting. I would recommend accepting it for publication after a minor revision
Line 12: add (E. gracilis) after Euglena gracilis
Line 30: replace Euglena gracilis by E. gracilis
Line 63: rewrite the objective 1
Line 39: low-cost substrates for the production value-added materials replace it with low-cost substrates to produce value-added materials
Line 90 and Line 105: same title
Line 208: change the number and title of the table it is not matching the content
Add error bars to the graphs and standard deviation to the results provided in the tables
Add the number of replicates as a footnote of the tables and figures
Line 223 Avoid discussion of the results in the results section
Remove line 239-242
334 Correct the number of figure 5
Line 346: kindly discuss the possibility of changing the pH under the same process, so the cultivation started at pH and after reaching an interesting biomass amount, the pH can be increased to 8 to increase the paramylon production
Line 396: reference #11 is missing the volume
ref # 17 need to be completed
Author Response
We, all the authors, appreciate the critical feedback from the editor and the reviewers. We have reviewed the entire manuscript carefully and wrote responses to all issues mentioned. All the mismatched or ambiguous description have revised. The answers have responded to all questions and comments raised by the reviewers. Thank you for your comments and suggestions.
Point 1: Line 12: add (E. gracilis) after Euglena gracilis.
Response 1: As commented, the phrase was revised to “Euglena gracilis (E. gracilis)” (line 12 in p.1).
Point 2: Line 30: replace Euglena gracilis by E. gracilis
Response 2: The word was revised to E. gracilis as commented (line 30 in p.1).
Point 3: Line 63: rewrite the objective 1
Response 3: As suggested, we revised the objective (1) to make it clearer (line 64-65 in p.2).
Point 4: Line 39: low-cost substrates for the production value-added materials replace it with low-cost substrates to produce value-added materials
Response 4: As suggested, the phrase was replaced to “low-cost substrates to produce value-added materials” (line 39 in p.1).
Point 5: Line 90 and Line 105: same title

Response 5: The subsection title was revised: 2.2.2. Experiment using different carbon concentrations (line 106 in p.3).
Point 6: Line 208: change the number and title of the table it is not matching the content. Add error bars to the graphs and standard deviation to the results provided in the tables. Add the number of replicates as a footnote of the tables and figures
Response 6: As suggested, the numeration of tables and figures were corrected to match with the body text. Also, we put the missing information on the number of replicates and standard deviation into the figures and tables.
Point 7: Line 223 Avoid discussion of the results in the results section
Response 7: As suggested, the sentences were revised to make them clear in order to describe the results only (line 236-237 in p.7).
Point 8: Remove line 239-242
Response 8: As commented, the sentences were deleted (line 255-258 in p.8).
Point 9: 334 Correct the number of figure 5
Response 9: The numbering of the figure was incorrect from the previous one and we have corrected.
Point 10: Line 346: kindly discuss the possibility of changing the pH under the same process, so the cultivation started at pH and after reaching an interesting biomass amount, the pH can be increased to 8 to increase the paramylon production
Response 10: We had measured the pH of the medium by day 6, but there were no drastic changes of pH values throughout the cultivation period in all conditions (regardless of the initial pH or the substrate type).
Point 11: Line 396: reference #11 is missing the volume
Response 11: As commented, the reference information was revised (line 417-418 in p.11).
Point 12: ref # 17 need to be completed
Response 12: As commented, the reference information was revised referring to the Author Guideline of the Applied Sciences (line 429-431 in p.11).
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript submitted by Kim and coworkers deals with the optimization of the obtention of biomass and paramylon from heterotrophic cultures of Euglena gracilis.
The authors propose the use of dry spent from tomato as a carbon source in heterotrophic axenic culture. I find the manuscript a scientific piece that can be of interest of Applied Sciences readers. Nevertheless, I think that the presentation of the culture conditions (initial inoculum, temperature, shaking, type of light intensity and cycle of dark/light used) must be clearly detailed in the manuscript.
How the authors measure paramylon concentration? It must be included into material and methods.
Author Response
We, all the authors, appreciate the critical feedback from the editor and the reviewers. We have reviewed the entire manuscript carefully and wrote responses to all issues mentioned. All the mismatched or ambiguous description have revised. The answers have responded to all questions and comments raised by the reviewers. Thank you for your comments and suggestions.
Point 1: The authors propose the use of dry spent from tomato as a carbon source in heterotrophic axenic culture. I find the manuscript a scientific piece that can be of interest of Applied Sciences readers. Nevertheless, I think that the presentation of the culture conditions (initial inoculum, temperature, shaking, type of light intensity and cycle of dark/light used) must be clearly detailed in the manuscript.
Response 1: As suggested by reviewer, the detailed information on light-dark cycle was added (line 79 in p.2 in p.2 and line 111-112 in p.3). Also, we addressed that the culture conditions for the experiments described in Section 2.2.1 and 2.2.2 are the same so allow the readers to refer (line 112 in p.3).
Point 2: How the authors measure paramylon concentration? It must be included into material and methods.
Response 2: As suggested by reviewer, more detailed information on the paramylon quantification were additionally described into material and methods section as follows (line 156-160 in p.4): “The lyophilized biomass was suspended in acetone and sonicated for cell disruption. After centrifugation, the pellet was resuspended in 1%(w/v) SDS, heated at 100 °C, and cooled to room temperature. The suspension was centrifuged and washed twice with distilled water. The final pellet was dried overnight at 60 °C prior to weighing.”
Author Response File: Author Response.docx
