Improvement of a Specific Culture Medium Based on Industrial Glucose for Carotenoid Production by Xanthophyllomyces dendrorhous

Round 1

Reviewer 1 Report

General comments:

 

The manuscript presents some data on carotenoids and biomass production in X. dendrorhous. Although the experiments are rather basic, some new results are provided.

 

English and grammar should be improved. There are numerous basic mistakes.

 

Materials and Methods:

It would be worth to explain why experiments done in 2L and in 110L reactors were not performed under the same conditions.

 

Results and Discussion:

 

In section 3.1, biomass growth is expressed in terms of OD, while it would be necessary to convert that to g/L, as this is more suitable for comparison with other research.

 

Standard deviations are shown in the Figures, and this should then also be provided everywhere in the text (e.g, Line 208 “… decreased 28%...”, etc.).

 

In section 3.2, please , exactly explain how the experiments without vitamins (CD medium) were performed. For example, in which medium was the preculture inoculum for such studies grown? Etc.

 

In section 3.3., the exact difference between reagent grade glucose and industrial grade glucose (composition, purity, etc.) should clearly be explained, considering that comparing their effect is the objective of this specific experiment.

How much fructose, maltose, etc. (and other compounds) were present?

 

The Discussion, line 281, is not correct. It is claimed that the presence of simple monosaccharides is industrial glucose is more favourable; while the opposite occurs (it contains non-monosaccharide sugars!).

 

The Discussion (in general) should also be improved. For example, the comments on top of page 9 (ref. 38, 39, 40), do not really help explaining the results observed in this study. The same is observe din other parts of the text/discussion. This should be improved.

 

Section 3.4: again, if standard deviations are provided in figures and tables, this should also be done in the text (e.g., Line 324 “increase of 50% …”, etc.).

 

Page 12: the authors explain the positive effect of adding glutamate, which they claim to be a possible strategy to increase yields. However, how expensive would the addition of glutamate be at large scale? Would this be suitable? This needs some a better discussion.

Also, can glutamate be used as an additional carbon/energy source by this organism? If this is the case, then the higher yield is probably due to the higher amount of substrate. This needs also some better discussion.

 

Conclusions:

 

Should be improved, among others based on the above comments.

 

References:

 

There is a rather high number of references (56) for a quite short manuscript. Are they all really useful?

Author Response

Reviewer 1

1. English and grammar should be improved. There are numerous basic mistakes.

The manuscript was submitted to the MDPI English proof-reading services, the evidence is included at the end of this document.

2. Materials and Methods:

It would be worth to explain why experiments done in 2L and in 110L reactors were not performed under the same conditions.

In section 2.5 (before line 178), the agitation difference between the 2 L and 110 L systems was specified. Now lines 186 to 189 “The difference in stirring speeds between the fermentation systems is due to that the 110 L bioreactor does not have the ability to stir at the same speed as the 2 L system, however, due to their geometric designs, both are capable to supply the oxygenation required under desired conditions. The bioreactors…”.

3. Results and Discussion:

In section 3.1, biomass growth is expressed in terms of OD, while it would be necessary to convert that to g/L, as this is more suitable for comparison with another research.

We thank the Reviewer for the kind suggestion; however, since the beginning of the experiments we only considered to express biomass growth as OD, as can be seen in section 3.1, Figures 2 and 3 where biomass growth is shown in Optical Density (OD) units. To modify the units of the graphs would take more than 10 days (Limit time to address the corrections) since nowadays we do not have access to our research institution due to the COVID outbreak, our institution remains closed. Furthermore, to change these units to grams per liter (g/L) since we would have to formulate other correlation experiments.

On the other hand, there are published reports showing O.D. as a method to determine the biomass growth of X. dendrorhous. Some examples are the articles by Jiang et al. 2020 (doi.org/10.1016/j.bej.2020.107519), Alcaino et al. 2016 (DOI 10.1371/journal.pone.0162838), Casteblanco-Matiz et al. 2015 (DOI 10.1007/s00203-015-1153-9), etc.

Standard deviations are shown in the Figures, and this should then also be provided everywhere in the text (e.g, Line 208 “… decreased 28%...”, etc.).

In section 3.1 (before line 208, now line 215), the results were expressed considering the standard deviations shown in the figures. Line 215 “27.76±1.0%”.

In section 3.2, please, exactly explain how the experiments without vitamins (CD medium) were performed. For example, in which medium was the preculture inoculum for such studies grown? Etc.

In section 3.2 (before lines 235 to 236), information was added to the document allowing the reader easily follow and understand the purpose of the developed study mentioned in line 242 of the manuscript. Lines 245 and 246 “(see section 2.3)”.

In the section 2.5 (before line 144), it was specified the operational condition for the vitamins study. Lines 145 to 168. “It was studied the impact on the cell growth and carotenoids production by X. dendrorhous when vitamins such as pantothenic acid, nicotinic acid, inositol, thiamine, pyridoxine, p-aminobenzoic acid, and biotin all together were added to the CD culture medium (high purity glucose obtained from Sigma-Aldrich). Similarly, the absence of vitamins in the CD culture medium was also evaluated. Additionally, to the previous assays industrial glucose was investigated as an alternative carbon source with the aim to replace high purity glucose; consequently, the presence/absence of vitamins was also evaluated in this culture medium.

An initial experiment was carried out with the CD culture medium [30]. It was used high purity glucose (reagent grade) considering i) the presence, and ii) the absence of vitamins at pH 6.0. The results were compared to obtain specific information on the way these micronutrients impact on biomass growth and total carotenoids production. An additional experiment was carried out, in which the CD culture medium was formulated using industrial grade corn glucose as a carbon source (obtained from ALMEX) instead of the high purity glucose. The purpose of this formulation was to obtain a lower cost culture medium for carotenoids production that can be scaled at pilot, and industrial levels. The difference between the purity of the substrate and the carotenoids production was evaluated. The operational conditions used for this study are described in section 2.2 and the inoculum used was previously activated in CD culture medium for 72 h at 20 ° C and 250 rpm.”

In section 3.3., the exact difference between reagent grade glucose and industrial grade glucose (composition, purity, etc.) should clearly be explained, considering that comparing their effect is the objective of this specific experiment. How much fructose, maltose, etc. (and other compounds) were present?

This is explained in section 2.3, lines 157 to 166, “It was used high purity glucose (reagent grade, section 2.2, Sigma-Aldrich) considering i) the presence, and ii) the absence of vitamins at pH 6.0. The results were compared to obtain specific information on the way these micronutrients impact on biomass growth and total carotenoids production. An additional experiment was carried out, in which the CD culture medium was formulated using industrial grade corn glucose as a carbon source (obtained from ALMEX) instead of the high purity glucose. The purpose of this formulation was to obtain a lower cost culture medium for carotenoids production that can be scaled at pilot, and industrial levels”.

In section 3.3, it was specified the composition of the sugars present in the industrial grade glucose. Before lines 279 to 281, now lines 286 to 289 “Industrial glucose is highly fermentable (Fig. 3), of its total composition, 72.5% are carbohydrates mainly as glucose (85-86%), containing also maltose (13.44%) and fructose (0.08%) [15,37], according to the specifications of the supplier”.

A complete characterization of industrial glucose is not available, only its sugar composition. This issue will be considered in a future publication.

The Discussion, line 281, is not correct. It is claimed that the presence of simple monosaccharides is industrial glucose is more favorable; while the opposite occurs (it contains non-monosaccharide sugars!).

In line 289 (before line 281), section 3.3, "presence of simple monosaccharides" was modified by " simple sugars (glucose and fructose) in conjunction with maltose ". We consider that with this modification it is better understood the purpose and discussion of employing an industrial carbon source with the presence primarily of glucose.

The Discussion (in general) should also be improved. For example, the comments on top of page 9 (ref. 38, 39, 40), do not really help explaining the results observed in this study. The same is observe din other parts of the text/discussion. This should be improved.

Lines 294 to 303, to support and reinforce the idea that yeast needs glucose (or some formulation mainly of glucose such as dextrose) as the main carbon source in order to carry out the production of biomass and/or carotenoids efficiently, the following paragraph was included in the manuscript:

“Nangia et al. [38] evaluated the impact of different carbon sources (rice, cane juice, and sucrose) on the production of astaxanthin by the yeast mutants of X. dendrorhous, and compared the results against the production obtained using dextrose as the main carbon source. The results showed yeast growth for all conditions, although the pigmentation varied from 1.5 to 2.5 OD units. Dextrose is used as a carbon source, as it is mainly assimilable for the production of biomass and astaxanthin, since it can be used directly in the metabolism of glycolysis, and through this route, in the biosynthesis of fatty acids and subsequently the biosynthesis of carotenoids, and astaxanthin [39,40]. However, glucose, in excessive amounts has been reported to be a repressor of astaxanthin synthesis by the so-called Crabtree effect [41]”.

Section 3.4: again, if standard deviations are provided in figures and tables, this should also be done in the text (e.g., Line 324 “increase of 50% …”, etc.).

In line 332 (before line 324), it was incorporated into the text, the standard deviation obtained for the carotenoids production between the comparison of treatments 1 and 2 of the experimental design “45.33±14.67%”.

Page 12: the authors explain the positive effect of adding glutamate, which they claim to be a possible strategy to increase yields. However, how expensive would the addition of glutamate be at large scale? Would this be suitable? This needs some a better discussion.

The conclusions clarified that the improved culture medium is less expensive than culture media such as YM or YPD with potential for scaling up the process. We also discussed that using food grade glutamate the fermentation process could reduce its cost even more. Before lines 444 to 447, now lines 454 to 458. “The low cost culture medium formulated in this study, has the advantage of being practical for scale up to pilot and even industrial levels for carotenoids and astaxanthin production using X. dendrorhous. Moreover, it has the capability to be evaluated in the cultivation of other species of yeasts and the cost could be reduced considerably more by using food grade glutamate”.

Also, can glutamate be used as an additional carbon/energy source by this organism? If this is the case, then the higher yield is probably due to the higher amount of substrate. This needs also some better discussion.

Discussion on the role of glutamate as an activator of carotenoid and astaxanthin biosynthesis in X. dendrorhous was improved. Before lines 369 to 374, now lines 379 to 384 “…activate genes that facilitates the assimilation of glucose, decreases the generation of reactive oxygen species, increases the channels of isocitrate flow in the glyoxylate cycle, and participates in the cycle of the five-carbon compounds promoting the accessibility of acetyl-CoA as precursor for carotenoids biosynthesis and shifts the metabolic flow to obtaining astaxanthin as the main product [19,20,27]”.

There is a rather high number of references (56) for a quite short manuscript. Are they all really useful?

We consider that all the 56 references cited are useful in the document, most of the define the sequence of the manuscript and provide additional information to the reader.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper present an interesting study regarding the possible replacement of culture media as YM and YPD with a low cost defined culture medium (CD) based on industrial glucose for carotenoid production by Xanthophyllomyces dendrorhous, with promising results.

 

The manuscript is well written, but some revisions are required:

In section 2.6 Analytical techniques more details must be given regarding the experimental protocol and apparatus used for HPLC determination of carotenoids produced by X. dendrorhous. Also, since the references indicated for the methods used in the evaluation of substrate consumption and of total carotenoids content aren’t open access, it would be useful to describe these protocols in more detail.

  In section Results, paragraph 2.3 supplementary results must be presented regarding the HPLC-MS/MS determination of endogenous hormones during seed germination, not only the determined content of those hormones. Mass spectra and chromatograms would be appropriate

Author Response

Reviewer 2

2. Materials and Methods:

In section 2.6 Analytical techniques more details must be given regarding the experimental protocol and apparatus used for HPLC determination of carotenoids produced by X. dendrorhous.

The experimental methodology for the determination of total carotenoids (section 2.6, lines 198 to 200) was carried out as described by Sedmak et al. 1990, and this was stated in the document as, “The total carotenoids content was quantified using the methodology described by Sedmak et al. [32]”.

The method uses small amounts of sample, it is fast, sensitive, and reproducible. It is based on the extraction of carotenoids using organic solvents, then their separation by polarity and subsequent quantification by spectrophotometry. Due to these characteristics, it was used as the main method of carotenoids quantification for the samples obtained from the present study. This former information was not included in the document since the reference describe it in detail. 

The HPLC analysis described in section 2.6 (lines 200 to 202), “It was corroborated by high-performance liquid chromatography (HPLC) that astaxanthin is the main carotenoid produced by the X. dendrorhous 25-2 strain used in the current study (supplementary Figure 1; Fig. S1).”, it was used to demonstrate the presence of astaxanthin as the main carotenoid produced by X. dendrorhous 25-2. However, this analysis was only performed for two samples of the present study, therefore the rest of the samples are reported according to what was described by Sedmak et al. 1990. Consequently, the analysis was presented as a supplementary figure (lines 462 to 467), “Figure S1: Disposition of astaxanthin as the major carotenoid produced by X. dendrorhous 25-2. A) Astaxanthin standard curve and B) final production chromatogram. For this determination, it was used a VARIAN® ProStar 220 HPLC and a VARIAN® ProStar 325 Ultraviolet-Visible detector. The mobile phase used was a hexane-acetone mixture in a ratio 82:18 (v/v) at a flow of 1.2 mL/min. The stationary phase was a Luna Phenomenex® column conditioned at 30 °C”.    

In section Results, paragraph 2.3 supplementary results must be presented regarding the HPLC-MS/MS determination of endogenous hormones during seed germination, not only the determined content of those hormones. Mass spectra and chromatograms would be appropriate.

We really appreciate your kind suggestion; however, to identify and quantify astaxanthin produced by HPLC-MS is really complex at this time since nowadays we do not have access to our research institution due to the COVID outbreak, our institution remains closed. In this sense, it will be no possible to perform HPLC determinations within the 10 days period to attend the manuscript corrections. This issue must be attended on future manuscript, where we will focus on astaxanthin production with an overproducing strain, that currently is under process of development. We humble state that there are publications that show the determination of total carotenoids expressed in mg/L without the need to report HPLC-MS/MS (Farías et al. 2018; doi.org/10.1016/j.bej.2018.04.016, Smidch et al. 2011; doi 10.1007/s00253-010 -2976-6).

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The comments were mostly adequately addressed.