Rhodotorula Strains Isolated from Seawater That Can Biotransform Raw Glycerol into Docosahexaenoic Acid (DHA) and Carotenoids for Animal Nutrition
Round 1
Reviewer 1 Report
The manuscript Rhodotorula strains isolated from seawater that can biotransform raw glycerol into docosahexaenoic acid (DHA) and carotenoids for animal nutrition concerns the isolation of red yeast from the environment, their identification and the study of metabolites. Research on enzymatic activity is interesting, and enrichment of seawater with glycerol as a culture medium is also worth research. However, the article requires some fine-tuning before it can be published.
Detailed comments:
line 29 - Rhodotorula should be in italics
line 44-45 - an attempt was made to cultivate Rhodotorula yeast in media containing glycerol as a carbon source, these were not from marine isolates, but the same are present in marine (e.g. DOI: 10.1007 / s11274-019-2732-8, DOI: 10.1016 / j.bcab.2020.101634, DOI: 10.1007 / s12010-019-03023-z)
line 90 - there should be an inoculum instead of inoculums
line 96 - why 10% glycerol?
Chapter 2.7 - what form were commercial kits made of?
Chapter 2.8 - Please describe the method in more detail
Chapter 2.9 - please describe the identification parameters in more detail, e.g. PCR, primers, the methodology should start from this chapter, since this is the first chapter in the results
line 372-383 - please compare the results to other Rhodotorula yeasts (not isolated from seawater) that were grown on glycerol-containing media (eg DOI: 10.1007 / s11274-019-2732-8, DOI: 10.1016 / j.bcab. 2020.101634, DOI: 10.1007 / s12010-019-03023-z)
line 465-467 - as mentioned above, glycerol is used to grow red yeast, many articles report such research
Author Response
Answer to Reviewer 1
1.- Reviewer: English language and style are fine/minor spell check required
Answer: English language has been improved
Comments and Suggestions for Authors
2.- Reviewer: The manuscript Rhodotorula strains isolated from seawater that can biotransform raw glycerol into docosahexaenoic acid (DHA) and carotenoids for animal nutrition concerns the isolation of red yeast from the environment, their identification and the study of metabolites. Research on enzymatic activity is interesting, and enrichment of seawater with glycerol as a culture medium is also worth research. However, the article requires some fine-tuning before it can be published.
Answer: ms has been improved with the reviewer suggestions.
Detailed comments
3.- Reviewer: line 29 - Rhodotorula should be in italics
Answer: Now Rhodotorula is in italics in line 30.
4.- Reviewer: line 44-45 - an attempt was made to cultivate Rhodotorula yeast in media containing glycerol as a carbon source, these were not from marine isolates, but the same are present in marine (e.g. DOI: 10.1007 / s11274-019-2732-8, DOI: 10.1016 / j.bcab.2020.101634, DOI: 10.1007 / s12010-019-03023-z)
Answer: The information has been incorporated as follow (lines 44–46): and Rhodotorula glutinis, R. mucilaginosa, and R. gracilis have been able to use glycerol and potato wastewater as a carbon source [6-8], but in low concentration of Sodium (≤ 1.1%).
5.- Reviewer: line 90 - there should be an inoculum instead of inoculums
Answer: The change has been done in line 129 in the new version of the ms.
6.- Reviewer: line 96 - why 10% glycerol?
Answer: Other studies for raw glycerol as a carbon source have used this concentration, around it or less in some cases (for example, Papanikolau et al. 2004, Posada & Cardona 2010, between), then we use this concentration as a reference. In the paper that the reviewer provides above, Kot et al. 2019 ([6] in the references of the manuscript) have been show that “The amount of glycerol added to media higher than 10% significantly decreased the growth rate of yeast”. Now, we have incorporated that data in the new version of the manuscript (line 398)
Papanikolaou, S., Fick, M., & Aggelis, G. (2004). The effect of raw glycerol concentration on the production of 1, 3‐propanediol by Clostridium butyricum. Journal of Chemical Technology & Biotechnology: International Research in Process, Environmental & Clean Technology, 79(11), 1189-1196.
Posada, J. A., & Cardona, C. A. (2010). Design and analysis of fuel ethanol production from raw glycerol. Energy, 35(12), 5286-5293.
7.- Reviewer: Chapter 2.7 - what form were commercial kits made of?
Answer: In the new version of the manuscript, the description of the methods area as follow (lines 239-243): The quantification of glucose, phosphorus and cholesterol were performed using commercial kits by the company Human Diagnostics (Wiesbaden, Germany), following the supplier’s instructions (Glucose liquicolor by GOD-POD method at 500 nm, Phos-phorus liquirapid by phosphomolybdic acid at 340 nm and Cholesterol liquicolor by CHOD-PAP method at 500nm).
8.- Reviewer: Chapter 2.8 - Please describe the method in more detail
Answer: In the new version of the manuscript, to descript these methods in more detail it has been added the following part (lines 258-265): The activity of L-malate dehydrogenase was assayed as it catalyzed the formation of malate from oxaloacetate. The assay medium contained 80 mM K2HPO4 buffer, pH 7.9, at 20°C, 0.1 mM NADH, 150 mM MgCl2 * 6H2O, and 0.2 mM oxaloacetate. Absorption was monitored at 340 nm following the addition of the supernatant. Activity of LDH was measured with a reaction mixture that contained 80 mM K2HPO4 buffer, pH 7.9, at 20 °C, 3.2 mM pyruvate and 0.1 mM NADH. Absorbance was monitored at 340 nm following the addition of the supernatant. MDH and LDH activity measurements were corrected for nonspecific NADH oxidation.
9.- Reviewer: Chapter 2.9 - please describe the identification parameters in more detail, e.g. PCR, primers, the methodology should start from this chapter, since this is the first chapter in the results
Answer: In the new version of the manuscript, to descript this method in more detail it has been added the sequence of primers (lines 90-97): “The amplification of the ribosomal gene 18s rRNA was carried out by PCR, using the primers described 18S1 (5‘-AACCTGGTTGATCCTGCCAGTA-3’) and 18S12 (5’-CCITGTTACGACITCACCTTCCTCT- 3’), [18]. Reaction mixture contained the fol-lowing reagents: 5X buffer of dNTPs, 2 mM of MgCl2, 1 U of Go-taq polymerase, 0.5 μM of each primers and approximately, 1.5 µL of DNA in a final volume of 25 µL. The amplification conditions used were specified by Honda et al. [18] as follow: initial denaturation at 95°C for 5 min, followed by 35 amplification cycles. Each cycle consisted of denaturation at 95°C for 30 s, alignment for 30 s at 55ºC and extension at 72°C for 1 min, with a final extension step of 72°C for 10 min.”
Note: This chapter has been moved to the beginning of the methodology as suggested by the reviewer (lines 85-122). Chapters and references have been renumbered.
10.- Reviewer: line 372-383 - please compare the results to other Rhodotorula yeasts (not isolated from seawater) that were grown on glycerol-containing media (eg DOI: 10.1007 / s11274-019-2732-8, DOI: 10.1016 / j.bcab. 2020.101634, DOI: 10.1007 / s12010-019-03023-z)
Answer: In the new version of the manuscript, the discussion of the information provided by the reviewer has been incorporated as follow (lines 396-401): “In non-marine Rhodotorula (R. glutinis, R. mucilaginosa and R. gracilis) have been showed that glycerol can be used to produce simultaneously lipids and carotenoids, nevertheless glycerol concentration higher than 10% can significatively decrease the growth rate [6-7]. R. gracilis seems to be a promising candidate for lipids and carotenoids production using low-cost wastes [8], with similar yields to the present study, but at low sodium concentration. In the present study, the marine origin of the strains used can be an advantage to cope with the osmotic stress compared with non-marine Rhodotorula.”
11.- Reviewer: line 465-467 - as mentioned above, glycerol is used to grow red yeast, many articles report such research
Answer: The information has been incorporated as follow (lines 44–46): and Rhodotorula glutinis, R. mucilaginosa, and R. gracilis have been able to use glycerol and potato wastewater as a carbon source [6-8], but in low concentration of Sodium (≤ 1.1%).
Reviewer 2 Report
Dear authors,
I congratulate you on the comprehensive work that you have made to present the results of an experimental study of achieving DHA from crude glycerol by means of Rhodotorula species. The experimental protocol is detailed, the results are well explained, the discussions are suitable and sustain the results. The conclusions of the work are well summarized. Anyway, I would still recommend you add some information about the raw glycerol that you used in the experimental design, in terms of its chemical profile. There is no specification about the percentage of glycerol content in biodiesel-derived raw glycerol fraction or about the presence of other chemicals (impurities perhaps) that might interfere with the development of the microorganisms and with the production of the metabolites (DHA especially).
Still, some minor changes are specified in the attached pdf document that I hope will help you to improve the content of your paper.
Good luck with the publication!
Sincerely,
Reviewer
Comments for author File: 
Comments.pdf
Author Response
Answer to Reviewer 2
1.- Reviewer: (x) Moderate English changes required
Answer: English language has been improved with the reviewer suggestions.
Comments and Suggestions for Authors
2.- Reviewer: Anyway, I would still recommend you add some information about the raw glycerol that you used in the experimental design, in terms of its chemical profile. There is no specification about the percentage of glycerol content in biodiesel-derived raw glycerol fraction or about the presence of other chemicals (impurities perhaps) that might interfere with the development of the microorganisms and with the production of the metabolites (DHA especially).
Answer: Unfortunately, we did not have the exact the percentage of glycerol content or the presence of other chemicals or impurities in the biodiesel-derived raw glycerol. Nevertheless, we used a highly concentrated fraction of glycerol obtained from fried food oil waste and in the new version of the manuscript, we have introduced the concept of “nominal concentration” in the materials and methods chapter after the concentration of raw glycerol (line 135). Also, we introduce a paragraph with the origin of the raw glycerol used as follow (lines 142-144): “In this study, the raw glycerol used, correspond to a highly concentrate fraction of glycerol collected during the industrial production of biodiesel from fried food waste oils (Comercial Verdemar Ltd Company, (COVEMAR-CHILE).”
Finally, with the objective of to know the effects of other chemicals or impurities that can be present in the raw glycerol and that might interfere with de production of metabolites of the assayed strains, we use analytical grade glycerol as a control.
3.- Reviewer: Still, some minor changes are specified in the attached pdf document that I hope will help you to improve the content of your paper.
Answer: we really appreciate and thanks the changes specified by the reviewer in the attached document. All of them have been used to improve the manuscript especially in the abstract. We also specified other studies where raw glycerol has been used to produce lipids by Rhodotorula (lines 44 to 46 in the new version of the manuscript). Finally, the expression of concentration of glycerol was explained in the answer above and we specified that in the line 135 as a “nominal concentration”.
Round 2
Reviewer 1 Report
The manuscript has been improved, no further comments.
