Strategies for Recovery, Purification and Quantification of Torularhodin Produced by Rhodotorula mucilaginosa Using Different Carbon Sources

Round 1

Reviewer 1 Report

This article attempts to explore the strategies for recovery, purification and quantification of Torularhodin synthesized by Rhodotorula mucilaginosa using three different carbon substrates. Although the study design is reasonable. There are a few things to consider especially when purification and quantification are concerned:

- Being a carotenoid, the compound is highly sensitive to oxidation and thermal decomposition. The acid treatment may also lead to a loss of carotenoids. For comparison, pure compound (maybe just beta-carotene) may be used as process control (for overall estimation). Also, how the author maintained the reduced environment during extraction?

- Regarding different substrates, the inoculum size itself was 1 g/L, then the xylose substrate seems too useless when feeding a very high substrate of 50 g/L. Also, residual substrate data should be provided in the main text for substrate conversion efficiency.

- Without such conversion data, the conclusion is misleading that glycerol and xylose can be utilized. Especially, the torularhodin content gets reduced.

- Whole genome submission ID and related data should be provided.

- For industrial production, the use of organic solvents is highly unsuitable (when the use is a pharmaceutical industry).

Overall the article's theme is good to study but lacks some minor components. The authors may be requested to revise the manuscript accordingly.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Zeng and co-workers (Strategies for recovery, purification and quantification of torularhodin produced by Rhodotorula mucilaginosa using different carbon sources) is certainly within the scope of Fermentation. In general the manuscript is well written, but some issues need to be addressed before the manuscript is accepted for publication:

-The information given in L19 (Abstract) is confusing. If the carotenoid content was ~121 ug/g of cells, and ~63% of torularhodin was recovered, the amount in parenthesis (~50 ug/g of cells) makes nonsense, needs revision.

-L70 “in various industrials” needs revision (“have wider industrial applications”?)

-L80-82, ref. [15] doesn’t seem to be a good reference for “Lignocellulosic biomass.....…..” as “the most abundant renewable resources on the planet”. Certainly there are more specific reviews!

-L98-99 “It was identified ….. and whole genome sequencing blasting with GenBank” Here more details are needed. Where the “whole genome sequencing” was deposited? What sequence from GenBank was used? What was the identity?

-L100 “yeast extract peptone dextrose (YPD) agar” needs to be described in details (amounts -g/L- of each component).

-L107 centrifugation details needs to be in “g”, not “rpm” (also lines 118, 153, 159, 163, 170, 172, 183…)

-L108 a “synthetic media” is mentioned, but its description is only detailed in lines 112-113. This needs revision.

-L130 “Followed 10 mL” needs revision.

-L168 also needs revision.

-L177 ‘sorbitol phosphate buffer” needs to be described in detail. In the same line, “density of 0.7 g/L.” of what?

-L180-181…. if the cells where disrupted, how cells where centrifuged to “reach a cell density of 15 g/L”?? Confusing, needs revision.

-L199 the “gradient elution” needs more details.

-L209 “chloroform and n-hexane/ethanol” should be “chloroform OR n-hexane/ethanol”

-L214 a “Figure 3” is mentioned, before Figures 1 and 2……. this needs revision.

-L239 needs revision.

-L247 “ An adverse result…” what are the authors trying to say?

-Figure 1B: the values placed in top of each column are no needed (the left scale shows that), but only the different letters indicating the significant differences among treatments. The same for Figure 2C. These values pollute the data in the figure…..

My final concern is that since the authors have used known methods to extract and characterize the carotenes in yeast, was is the novelty/importance of the study? I would suggest that the authors need to compare their results with other papers describing the production/extraction of carotenes (including torularhodin) from R. mucilaginosa (e.g. Process. Biochem. 40:2985–2991, 2005; Methods Mol. Biol. 898:275-83, 2012; J. Taiwan Inst. Chem. Eng. 61:270–275, 2016; Biotechnol. Rep. 25:e00407, 2019), as many of these papers also used cheap agro-industrial substrates!

Author Response

Please see the attachment.

Author Response File: Author Response.pdf