Cutaneotrichosporon oleaginosus: A Versatile Whole-Cell Biocatalyst for the Production of Single-Cell Oil from Agro-Industrial Wastes
Round 1
Reviewer 1 Report
The authors in their review article described Cutaneotrichosporon oleaginosus, an oleaginous yeast species capable of synthesizing high amounts of lipid from different agro-industrial wastes. The possibilities of lipid synthesis in media with acetic acid, volatile fatty acids, crude glycerol, N-acetylglucosamine, lignocellulosic materials and wastepaper were described.
In my opinion, it would be worth describing the possibility of using genetic manipulation to modify these yeasts in order to obtain larger amounts of lipids or lipids of a different, more valuable composition.
Minor suggestion: Please change the enumeration of sections. Section 7. should be section 4, and conclusions should start with 5. not 8.
Author Response
Response to Reviewer 1 Comments
The authors in their review article described Cutaneotrichosporon oleaginosus, an oleaginous yeast species capable of synthesizing high amounts of lipid from different agro-industrial wastes. The possibilities of lipid synthesis in media with acetic acid, volatile fatty acids, crude glycerol, N-acetylglucosamine, lignocellulosic materials and wastepaper were described.
Point 1: In my opinion, it would be worth describing the possibility of using genetic manipulation to modify these yeasts in order to obtain larger amounts of lipids or lipids of a different, more valuable composition.
Response 1: We thank the reviewer for his/her suggestion because it enabled us to improve the quality of the manuscript. According to the reviewer’s comment, the manuscript has been revised as follows:
Pag. 30: “Actually, the biochemical pathways for oil accumulation in oleaginous yeasts are known and this favours the research work on finding low-cost and alternative feedstocks for SCOs production and on improving the bio-oil productivity by genetic tools or optimising the bioconversion process. Moreover, the deep knowledge of the genome of C. oleaginosus[34] allows the development of genetic engineering tools for enhancing the catalytic performance of this yeast by increasing the lipid production or obtaining a more valuable oil composition [134]. There are three main metabolic engineering approaches to improve the production of triacylglycerols in oleaginous yeasts: (1) the increase of lipids productivity by increasing the metabolic flow from sugars to lipids; (2) the re-design of the stoichiometry of lipids production for a more efficient conversion of carbon atoms and electrons to triglycerides; (3) the removal of biochemical pathways related to the synthesis of by-products that affect the lipids production, such as secondary metabolites, glycogen and degradation pathways of fatty acids [135-139]. Up to now, in the literature only two studies investigated different metabolic engineering approaches for C. oleaginosus ATCC 20509 [140,141].”
Point 2:Minor suggestion: Please change the enumeration of sections. Section 7. should be section 4, and conclusions should start with 5. not 8.


Response 2:The enumeration of sections has been revised according to the reviewer’s comment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Review to the manuscript catalysts-1442719 entitled “Cutaneotrichosporon oleaginosus: a versatile whole-cell biocatalyst for the production of single cell oil from agro-industrial wastes” by Nicola Di Fidio et al. submitted to Catalysts.
The focus of the review is non-conventional yeast Cutaneotrichosporon oleaginosus and its ability of the oil production from various substrates. C. oleaginosus has several technological advantages, incl. its wide substrate spectrum, high lipid yield and robustness which makes the yeast species promising industrial catalyst to convert agro-industrial wastes to microbial oil. In the manuscript detailed information is given on biorefinery approaches involving C. oleaginosus as the whole-cell catalyst and it summarizes state-of-art information on process reaction conditions, catalyst efficiency, oil production and its yield, and lipid profiles from substrates. Application of oleaginous yeasts for lipid production is a powerful method that can open new and sustainable avenues to valorise organic waste and produce valuable chemicals and biofuel. Therefore, the review article is on a very relevant subject. The subject of the review is in accordance with the aims and scope of Catalysts.
General remarks:
The manuscript is adequately structured and composed, finely detailed and clearly presented with comprehensive explanations given. It can be considered a rather comprehensive review of the state-of-the-art in applying C. oleaginosus to oil production. The explanations are thorough some of the information seems to be even excessively detailed which is making the manuscript long and diffuse in the chapters 3.1-3.5. It could enhance the readability of some subchapters instead of summarizing papers one by one the authors would resume and discuss the results in more concentrated way. Especially because the numerical data (yields, conditions etc) is given in tables, it is not always necessary to repeat it in the text.
The manuscript is written in an academic style and correct English language is used.
It would be beneficial for readers of the manuscript if it contains a scheme that is summarizing the main substrates and products covered in the review. It can be added as graphical abstract or Figure 1.
Specific comments:
Some of the used abbreviations have not been explained (lines 126, 656).
The title 3.1 (line 168) should in addition to acetic acid contain „volatile organic acids” as they are mentioned as suitable substrates in the subchapter.
Lines 373-381. Methanol is antibacterial substance but it should be mentioned that methylotrophic yeasts such as Ogataea polymorpha or Pichia methanolica could still significantly proliferate in the presence of methanol.
Lines 305, 466, 657, It is not recommended to use phrases like “performance was better/worse” which are from conversational English and not very academic style. Instead “performance increased/decreased” could be used.
Lines 486, 488, 528, 761, 1028, Table 4. N in N-acetylglucosamine and other amines should be in italics.
Table 5 and 6 could appear in the Supplementary Materials as it is not summarizing the lipid production properties of C. oleaginosus.
The chapter 7. Applications of single cell oils, strengths and weaknesses and 8. Conclusions should be renumbered following the chapter order.
Author Response
Review to the manuscript catalysts-1442719 entitled “Cutaneotrichosporon oleaginosus: a versatile whole-cell biocatalyst for the production of single cell oil from agro-industrial wastes” by Nicola Di Fidio et al. submitted to Catalysts.
The focus of the review is non-conventional yeast Cutaneotrichosporon oleaginosus and its ability of the oil production from various substrates. C. oleaginosus has several technological advantages, incl. its wide substrate spectrum, high lipid yield and robustness which makes the yeast species promising industrial catalyst to convert agro-industrial wastes to microbial oil. In the manuscript detailed information is given on biorefinery approaches involving C. oleaginosus as the whole-cell catalyst and it summarizes state-of-art information on process reaction conditions, catalyst efficiency, oil production and its yield, and lipid profiles from substrates. Application of oleaginous yeasts for lipid production is a powerful method that can open new and sustainable avenues to valorise organic waste and produce valuable chemicals and biofuel. Therefore, the review article is on a very relevant subject. The subject of the review is in accordance with the aims and scope of Catalysts.
General remarks:
Point 1:The manuscript is adequately structured and composed, finely detailed and clearly presented with comprehensive explanations given. It can be considered a rather comprehensive review of the state-of-the-art in applying C. oleaginosus to oil production. The explanations are thorough some of the information seems to be even excessively detailed which is making the manuscript long and diffuse in the chapters 3.1-3.5. It could enhance the readability of some subchapters instead of summarizing papers one by one the authors would resume and discuss the results in more concentrated way. Especially because the numerical data (yields, conditions etc) is given in tables, it is not always necessary to repeat it in the text.
Response 1: We thank the reviewer for his/her comments and suggestions because it enabled us to improve the quality of the manuscript. According to the reviewer’s comment, in order to enhance the readability of subchapters 3.1-3.5 a more concentrated discussion of the papers was adopted in the text. Moreover, numerical data reported in the tables were removed by the text.
Point 2:The manuscript is written in an academic style and correct English language is used.
Response 2: We thank the reviewer for his/her comment.
Point 3:It would be beneficial for readers of the manuscript if it contains a scheme that is summarizing the main substrates and products covered in the review. It can be added as graphical abstract or Figure 1.
Response 3: According to the reviewer, we added in the Introduction section a figure (Figure 1) summarising all the waste substrates converted to single cell oil by C. oleaginosus and described in the present review. The manuscript has been revised as follows:
Pag. 3:“For this reason, similarly to other review works related to other species of oleaginous yeasts [36], the present review summarises for the first time recent biorefinery approaches involving the whole-cell biocatalyst C. oleaginosus (Fig. 1).
Figure 1. Schematic representation of the main substrates and products covered in the review.”
Specific comments:
Point 4:Some of the used abbreviations have not been explained (lines 126, 656).
Response 4: The abbreviations indicated by the reviewer has been explained, as follows:
Pag. 3: “This review was based on a phylogenetic analysis of seven markers, more precisely large subunit (LSU) ribosomal RNA (domains D1/D2), small subunit (SSU) ribosomal RNA, Internal Transcribed Spacer (ITS), genes encoding the proteins RPB1, RPB2, TEF1, CYTB as well as a combination of morphological, biochemical and physiological characteristics [50].”
Pag. 19: “As reported in Table 5, no growth was observed on the undetoxified cardoon hydrolysate due to the synergistic toxic effect of various by-products produced during the steam explosion pretreatment, such as acetic acid (2.1 g/L), 5-hydroxymethylfurfural (5-HMF,0.32 g/L), furfural (0.42 g/L) and total phenolic compounds (5 g/L).”
Point 5:The title 3.1 (line 168) should in addition to acetic acid contain „volatile organic acids” as they are mentioned as suitable substrates in the subchapter.
Response 5:The title of the subsection 3.1 has been revised according to the reviewer’s comment, as follows:
Pag. 4: “3.1. Acetic acid and volatile organic acids”
Point 6:Lines 373-381. Methanol is antibacterial substance but it should be mentioned that methylotrophic yeasts such as Ogataea polymorpha or Pichia methanolica could still significantly proliferate in the presence of methanol.
Response 6:We agreed with the reviewer and the manuscript has been revised as follows:
Pag.11: “It has been demonstrated that methanol can be used, within certain threshold concentrations, to selectively inhibit the growth of contaminating microorganisms, such as bacteria, which can contaminate fermentation and limit the oleaginous yeast biomass production [76]. This strategy can be used when the fermentation medium and fermenter are not sterilised [82], even if some methylotrophic yeasts such as Hansenula polymorpha (now classified as Ogataea polymorpha) or Pichia methanolica can proliferate in the presence of methanol [83,84].”
Point 7:Lines 305, 466, 657, It is not recommended to use phrases like “performance was better/worse” which are from conversational English and not very academic style. Instead “performance increased/decreased” could be used.
Response 7:The English revision of the text was performed according to the reviewer’s suggestions.
Point 8:Lines 486, 488, 528, 761, 1028, Table 4. N in N-acetylglucosamine and other amines should be in italics.
Response 8:N in N-acetylglucosamine and other amines in the manuscript has been revised in italics.
Point 9:Table 5 and 6 could appear in the Supplementary Materials as it is not summarizing the lipid production properties of C. oleaginosus.
Response 9:According to the reviewer’s suggestion, Tables 5 and 6 (now Tables S1 and S2) have been shifted to the Supplementary Materials and the manuscript has been revised accordingly.
Point 10:The chapter 7. Applications of single cell oils, strengths and weaknesses and 8. Conclusions should be renumbered following the chapter order.
Response 10:The enumeration of sections has been revised according to the reviewer’s comment.
Author Response File: Author Response.pdf
