Simultaneous Lipid and Carotenoid Production via Rhodotorula paludigena CM33 Using Crude Glycerol as the Main Substrate: Pilot-Scale Experiments
, Pailin Boontawan 1, Pasama Boonyanan 2, Mariena Ketudat-Cairns 1 and Apichat Boontawan 1,3,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this manuscript, authors showed the simultaneous lipid and carotenoids production by Rhodotorula paludigena CM33 using crude glycerol as the main substrate. The manuscript may have potential interest. However, it doesn't seem like a very good fit for the Int. J. Mol. Sci. since the manuscript focuses on optimization of the culture conditions and fermentation. The manuscript was not well written. Several major comments must be addressed before the publication.
Major comments:
1. The title of the manuscript is simultaneous lipid and carotenoids production by Rhodotorula paludigena CM33. However, most of the Introduction part focus on the carotenoids. And the last word of the Introduction is “The aim of this study was to pave a way for the commercial production of carotenoids from oleaginous yeast using agro-industrial by-products”, which is not consistent with the title. I think that if the authors want to keep the current title, the Introduction part should contain more content of lipid production and show the motivation of this study about simultaneous lipid and carotenoids production.
2. The manuscript should contain a figure which shows the metabolic pathway of producing lipid and carotenoids simultaneously from glycerol of Rhodotorula paludigena CM33.
3. Line 142-147, the authors said that 40 g/L of crude glycerol was optimal and other studies had similar results with 40 g/L of pure glycerol as the optimal concentration. However, crude glycerol contained only 58% glycerol, so 40 g/L of crude glycerol is not similar with 40 g/L of pure glycerol.
4. Figure 2, the symbols which represent the glycerol, lipid, biomass and carotenoids should be shown in each of A, B and C. However, the authors just showed the symbols in Fig. 2A.
5. Some fold changes data in section 2.3 are confusing and difficult to follow, such lines 199-203 and 214-216. The authors should go through this part carefully and make it clear to whom the fold changes were being compared.
6. Table 3, the data of Repeated-batch were not showed appropriately. The authors should adjust the format of the table.
7. Line 210, do the authors mean 713 mg/L is the highest value among all the studies so far?
8. What’s the purpose of the Table 5? Do the authors want to compare with other studies and show the advantages of this study for producing the carotenoids and lipid? If so, I think the authors should add more discussion in the text.
9. Lines 233-235, the authors should add a reference here.
10. Lines 279-280, I don’t think I can follow this sentence. Can Figure 4b show something about the characteristics of the compound samples after undergoing the Preparative HPLC process?
11. Figure 6b, the text of x and y axis are not clear. The authors should provide high quality figures.
12. There are so many figures and tables in the main text which make the manuscript long and messy. Some tables and figures should be put into the SI, such as tables 4, 5, 6, 7 and figure 7.
Minor comments:
The manuscript was not well written. The authors should carefully check the writing.
1. Lines 17-18, the sentence should be rewritten.
2. Line 39, “glycerol [3-5].” should be “glycerol [3-5],”
3. Lines 47-48, the sentence should be rewritten.
4. Line 86, “to contained” should be “to contain”.
5. Lines 93-94, the authors should rewrite these sentences to combine the two references into one sentence.
6. line 187-188, the sentence should be rewritten.
7. ling 192, “could decreased” should be “could be decreased”
8. The legend of the Figure 4 should be “Microscopic images of R. paludigena CM33 cells (a) after the 1st pass, (b) after the 2nd pass, (c) after the 3rd pass, and (d) after the 4th pass using HPH”.
9. The strain names should be italic. The authors should go through the manuscript and correct all of them.
Comments on the Quality of English LanguageExtensive editing of English language required.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors provided their original research article dealing with lipid and carotenoids production with the use of Rhodotorula paludigena CM33 in media with crude glycerol. During the experiments, the authors applied Response Surface Methodology for simultaneous carotenoids and lipid production, as well as, different fermentation strategies, including batch, repeated batch, and fed-batch cultures in a 500 L bioreactor.
In my humble opinion, the manuscript has been prepared in a clear and concise way. Only some minor suggestions are provided below:
- The major flaw of the submitted manuscript:
Both in the title of the work and in the further text, the authors refer to, in addition to carotenoids, lipids production by this yeast. With such a detailed focus on the carotene and its derivatives, their preparation, purification, etc., the authors completely ignore the composition of the obtained lipids. Were chromatographic analyses performed to determine the fatty acid profile during the research? It seems to me that this should be a key element, next to knowing the composition of carotenoid pigments. I believe that the GC profile of fatty acids should be provided for at least the best-optimized cultures.
Furthermore, does the statement that there is a simultaneous production of these metabolites mean that the production of lipids and carotenoids is correlated with each other, and whether the same mechanism is responsible for the production of these secondary metabolites? Therefore, please add in the text about the production of these metabolites, i.e., lipids and carotenoids, in yeasts of the genus Rhodotorula.
- In the case of the aim of the work, some kind of statement of novelty may be provided to indicate the need for research on this issue.
- Table 5 was mentioned on page 4 for the first time, but the authors placed this table on pages 13-14. Thus, the table should be transferred and for the other tables, the authors should change the numbers.
- The reference list should be improved according to the Instructions for authors.
- The authors should check the italics for microorganisms names in the whole paper.
- In the case of Figure 2, can the authors use different colors for the data instead of markings in the form of geometric figures? I believe this will improve the visibility of this data.
- The conclusion part may be improved. Future perspectives may be provided.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript is prepared at a high scientific level. The presented results are very promising. Conducting experiments on a big scale in the second phase should be pointed out.
In my opinion, the manuscript has some lacks that should be corrected:
Line 315: "and incubated at 30 ºC overnight on a rotary shaker at 150 rpm for 48 h" overnight or for two days?
Please add more details about the method of crude glycerol quantitative determination.
Please add more details about the method of crude glycerol quantitative determination. Not only the name of the equipment and producer but also information about used the analytical parameters.
The yeast cells were harvested by using a centrifuge. Information about technological parameters should be added.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed most of my comments.
I just have two minor comments
1. Figure 3 seems to have a bad resolution. A high-quality figure needs to be provided.
2. For the responses of my comment 3, the authors said that they pretreated the crude glycerol before the experiments, and they used the pretreated and purified crude glycerol for the experiments. The author also analyzed the concentration of the glycerol after the pretreatment using HPLC. So, what’s the concentration of the glycerol in the crude glycerol after the pretreatment? I think this is necessary to show. Because this is the real concentration of glycerol in the crude glycerol used for the experiments. The results of the Section 2.1 showed that the crude glycerol used in this study was found to contain 58% glycerol. But this is not the real content of glycerol in the crude glycerol used for the experiments, right?
Author Response
Dear Editor,
I am submitting the response to the reviewer's comments file. There are 2 comments from the reviewer, 1. I increased the quality of Figure 3, and 2. I added the concentration of crude glycerol in section 2.1.
Author Response File: Author Response.pdf
