Light Induction of Seed Culture Accelerates Lutein Accumulation in Heterotrophic Fermentation of Chlorella protothecoides CS-41

Round 1

Reviewer 1 Report

In this work, the Authors provided experimental evidence for the efficiency of a new cultivation strategy to enhance the production of lutein in Chlorella protothecoides. To validate the proposed strategy, the Authors assessed several physiological and biochemical parameters, including the analysis of key metabolites, the evaluation of the expression profile of genes involved in carotenoid biosynthesis, central carbon metabolism, and photosynthesis.

The study is well conducted, and results clearly described, providing useful information for an improved lutein production from C. protothecoides.

There are some minor points to be addressed, as follows:

Page 3, line 128. Please indicate the temperature at which the mixture was heated. Also add information for the ELIASA.

Page 4, line 157. Add information how the cells were disrupted, if any equipment has been used to aid cell rupture.

Page 4, line 183. Add information about the internal standard used.

Add information about the equipment used to determine photochemical efficiency that are reported in the result section, although there is no mention in Material and Methods.

Figure 1, panel (f): the bar for the C18:2 fatty acid in high light conditions is not visible. Please check.

Figure 4, panel (b): figures are barely visible. I encourage the Authors to explore possible solutions to improve the readability of the figure.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Editor,

I am writing regarding the manuscript "Light induction of seed culture accelerates lutein accumulation in heterotrophic fermentation of Chlorella protothecoides CS-41" which I reviewed for Fermentation. I recommend accepting this paper after minor revisions.

The authors have demonstrated a novel cultivation strategy involving low light induction of seed cultures to boost lutein productivity during subsequent heterotrophic fermentation of the microalgae Chlorella protothecoides CS-41.

The paper is well-written and the claims are supported by doing the following recommendations

·       The abstract would benefit from explicitly stating the improvements in lutein content, yield and productivity achieved.

·       In the abstract, rephrase "Lutein has gained attention not only for its antioxidation properties but also for its potential anticancer effects..." to "Lutein has gained attention not only for its antioxidant properties but also for its potential anticancer effects..."

·       In the introduction, reword "They possess the ability to accumulate various nutritional products such lipids, proteins, and pigments..." to "They possess the ability to accumulate various nutritional products such as lipids, proteins, and pigments..."

·       In Section 2.1.1, change "The cultures are cultivated under three cultivation conditions:" to "The cultures were cultivated under three conditions:"

·       In Section 2.5, change "Out of the 26 analyzed metabolites, 24 exhibited significant differences." to "Out of the 26 analyzed metabolites, 24 showed significant differences."

·       Some discussion of scale-up challenges and economic feasibility analysis would make the case stronger for industrial adoption.

·       the abbreviations HD, LD, and DD used for the high light, low light, and dark conditions in this paper are not the most appropriate or clear. Use more descriptive abbreviations:High light (HL), Low light (LL), Dark (DK)

·       In Figure 1, legend should read "Post-hoc comparisons, different numbers of asterisks are used to indicate significant differences."

·       In Figure 2 legend, change "pathway analysis using" to "pathway analysis was performed using"

·       Add asterisks above the columns in Figures 3D, and 3F to denote statistically significant differences (p<0.05) between the three cultivation strategies based on the post-hoc comparison results.

·       In Figure 3 legend, change "different numbers of asterisks are used to indicate significant differences" to "asterisks indicate significant differences"

·       Standardize capitalization of metabolite names like glycerate vs Glycerate.

·       In conclusions, clearly specify limitations of the current study and note that further validation is still needed in scaled systems.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf