Enhancing Nutritional Value and Sensory Quality of Spirulina (Arthrospira platensis) Through Preharvest Co-Cultivation with Yeast Saccharomyces cerevisiae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents findings related to a potentially interesting approach. However, several critical points and inconsistencies within the Results and Discussion section require revision and clarification to enhance scientific rigor and clarity:

• Although commercially known as 'Spirulina,' recent taxonomic and genomic analyses have clarified that Arthrospira and Spirulina are distinct genera with substantial genomic and ecological differences. Therefore, the correct genus name for the commercially available phycocyanin source is Limnospira (formerly Arthrospira) (as reported here https://www.sciencedirect.com/science/article/pii/S2665927125001728?via%3Dihub).
• The authors highlight a significant increase in biomass production at yeast/microalgae ratios of 1:1000 and 10:1000. However, there is insufficient discussion of possible metabolic interactions or competitive mechanisms occurring at the higher ratio of 100:1000 that lead to the decrease in biomass. Please clarify.
• From Figure 1 i see that the Arthrospira platensis used is completely linearized, can this affect the growth performance? Why this morphology?
• Adjust the legend for Figure 1 since it is not specified the four images for the microscopic morphology.
• The kinetics of dissolved oxygen described are somewhat speculative. The interpretation regarding intensified respiration and photosynthesis requires additional evidence of established literature to confirm the hypothesized metabolic shifts during different incubation periods.
• Describe better the team of panelist used in the study. Where the sensory analysis conducted in sensory booths?
• The statement regarding consumer preference (lines 117-118) appears anecdotal without a supporting sensory or consumer study to substantiate it.
• The volatile compound profile is extensive, however, the authors inadequately differentiate between biological significance and mere analytical presence.
  For instance, numerous ketones and aldehydes are reported, but a clear explanation regarding their practical impact or thresholds relevant to human sensory perception is lacking 

• Although gene expression levels were measured, a direct correlation between transcript abundance, protein/enzyme activity, and observed biochemical changes (in β-carotene, linoleic acid, γ-linolenic acid) is lacking.

• There is no clear evidence presented that supports the claim that acetate specifically modulates gene expression directly. 

• Line 233 (Figure 3 legend): The p-values are mistakenly represented as ***P < 0.001, **P < 0.005, *P < 0.01, which does not follow standard statistical convention (commonly: *P<0.05, **P<0.01, ***P<0.001).

• Figure 2 legend: Units for pigment content (mg/kg or mg/g wet biomass) are inconsistent or unclear. Consistent units across the manuscript are necessary for clarity.

Comments on the Quality of English Language

Minor revision on English

Author Response

Comments 1: Although commercially known as 'Spirulina,' recent taxonomic and genomic analyses have clarified that Arthrospira and Spirulina are distinct genera with substantial genomic and ecological differences. Therefore, the correct genus name for the commercially available phycocyanin source is Limnospira (formerly Arthrospira) (as reported here https://www.sciencedirect.com/science/article/pii/S2665927125001728?via%3Dihub).

Response 1: Thank you for your careful attention to the taxonomic classification of the commercial phycocyanin source. We acknowledge the evolving understanding of Arthrospira's systematic position, as highlighted by recent genomic studies. However, we respectfully retain the term Arthrospira in our manuscript for the following reasons: While Limnospira has been proposed as a replacement for Arthrospira, this reclassification remains controversial and is not yet universally adopted in the phycocyanin research community. Notably, major databases (e.g., NCBI Taxonomy, AlgaeBase) still list Arthrospira as valid for these strains. In addition, retaining Arthrospira ensures direct comparability with prior studies and avoids introducing unnecessary confusion for readers familiar with the established nomenclature.

We have added a clarifying statement in the revised manuscript (see Line 310-312):
“Arthrospira remains the taxonomically validated classification per phylogenomic evidence (current study), whereas 'Limnospira' remains a contentious proposal lacking consensus.”.

 

Comments 2: The authors highlight a significant increase in biomass production at yeast/microalgae ratios of 1:1000 and 10:1000. However, there is insufficient discussion of possible metabolic interactions or competitive mechanisms occurring at the higher ratio of 100:1000 that lead to the decrease in biomass. Please clarify.

Response 2:

Thank you for your valuable comment. We agree that the biomass decrease at the 100:1000 ratio warrants further discussion. While our current data clearly show A. platensis survival stress (evidenced by biomass reduction, and microscopic observations), the underlying mechanisms remain unclear due to two key limitations: (1) the extreme yeast dominance (100:1000) creates a physiologically stressful environment that complicates mechanistic dissection, and (2) no prior studies have characterized such imbalanced interactions, leaving no direct literature precedents for comparison. To address this, we have added a paragraph to the Discussion explicitly stating these limitations and proposing future multi-omics studies to investigate yeast-microalgae interactions under extreme ratios.

We have added a clarifying statement in the revised manuscript (see Line 85-88):

“The biomass decline observed at a 100:1000 ratio was primarily attributed to survival stress in A. platensis; however, the underlying mechanisms remain incompletely elucidated due to the paucity of comparative studies.”

 

Comments 3: From Figure 1 i see that the Arthrospira platensis used is completely linearized, can this affect the growth performance? Why this morphology? Adjust the legend for Figure 1 since it is not specified the four images for the microscopic morphology.

Response 3: Thank you for your keen observation regarding the morphology of Arthrospira platensis (FACHB-902) in Figure 1. The linear morphology of A. platensis (FACHB-902) is a certified characteristic from the Freshwater Algae Culture Collection (FACHB), specifically selected for stable growth under controlled conditions. In aquaculture, this “linear” filamentous phenotype (vs. compact morphotypes prone to filtration issues) is preferred for optimal biomass production. Our data confirm this morphology does not impair growth, ensuring its suitability for studying yeast-microalgae interactions. We have revised the legend for Figure 1 to clearly label and specify the four microscopic images showing the morphological characteristics of Arthrospira platensis (FACHB-902).

We have added a clarifying statement in the revised manuscript (see Line 92-93):

Microscopic morphology of A. platensis: (1) monoculture; (2) yeast/microalgae ratio 1:1000;

 

Comments 4: The kinetics of dissolved oxygen described are somewhat speculative. The interpretation regarding intensified respiration and photosynthesis requires additional evidence of established literature to confirm the hypothesized metabolic shifts during different incubation periods.

Response 4: We appreciate your insightful comment. Based on the co-culture system's dynamics, we replaced speculative descriptions with mechanistic analysis supported by established literature, anchoring DO kinetics to glucose-driven respiration (0-12 h) and algal photosynthesis (12-24 h) in the revised manuscript (see Line 110-115):

This DO pattern was driven by metabolic shifts: (1) Rapid respiration during the initial phase (0-12 h), fueled by S. cerevisiae proliferation under glucose supplementation and phototrophic oxygen release; (2) DO recovery in the subsequent phase (12-24 h), mediated by A. platensis as the dominant photoautotroph, with biomass increase (Figure 1e) restoring oxygen production following glucose depletion.

 

Comments 5: Describe better the team of panelist used in the study. Where the sensory analysis conducted in sensory booths? The statement regarding consumer preference (lines 117-118) appears anecdotal without a supporting sensory or consumer study to substantiate it.

Response 5: Thank you for your comments. We confirm that the sensory evaluation was conducted in a standardized odor analysis room (Line 375-377). To substantiate the consumer preference statement, we have added a citation from Food Color and Appearance in Perspective to highlight that lighter biomass coloration enhances visual appeal by associating with purity, naturalness, and appetite stimulation [13,14] (Line 124).

Sensory evaluation of volatile flavor was conducted in a standardized sensory booth (ISO 8589:2007) under controlled lighting and ventilation conditions.

 

Comments 6: The volatile compound profile is extensive. However, the authors inadequately differentiate between biological significance and mere analytical presence. For instance, numerous ketones and aldehydes are reported, but a clear explanation regarding their practical impact or thresholds relevant to human sensory perception is lacking

Response 6: Thank you for your insightful comment. We agree and have added sensory thresholds and OAV values of norisoprenoids (the most abundant and flavor-significant compounds in this study) (Table S3) to contextualize their biological relevance, along with a discussion linking aldehydes to observed sensory attributes (Lines 178-181).

Notably, quantitative analysis of OAVs (Table S3) demonstrated that β-ionone, epoxy-β-ionone, (E)-4-oxo-β-ionone, β-cyclocitral, safranal, and dihydroactinidiolide significantly exceeded thresholds, underscoring their dominant role in the enhanced floral and fruity aromas of co-cultured microalgae.

 

Comments 7: Although gene expression levels were measured, a direct correlation between transcript abundance, protein/enzyme activity, and observed biochemical changes (in β-carotene, linoleic acid, γ-linolenic acid) is lacking. There is no clear evidence presented that supports the claim that acetate specifically modulates gene expression directly.

Response 7: Thank you for your insightful comment. We acknowledge the absence of direct enzyme activity measurements and specific evidence for acetate-mediated transcriptional regulation. The capacity of acetate supplementation to enhance carotenoid content has been previously demonstrated (Improving the growth of Spirulina in CO₂ absorption and microalgae conversion (CAMC) system through mixotrophic cultivation: Reveal of metabolomics). In addition, direct enzyme activity measurements were unattainable due to the inseparable nature of the two mixed microbial populations in the co-culture system. Arthrospira platensis FACHB-902, a non-model cyanobacterium, was subjected to comprehensive genome and transcriptome sequencing (datasets provided in Supplementary Files) to facilitate pathway enrichment analysis. These analyses identified enriched pathways involved in β-carotene, linoleic acid, and γ-linolenic acid biosynthesis. Gene expression levels were quantified by qPCR using primers designed based on genome-annotated enzyme sequences. Owing to technical challenges in isolating microalgal enzymes within the co-culture mixture, gene expression data provided the most reliable indication of metabolic shifts. Acetate, a predominant yeast-secreted metabolite, exhibited a positive correlation with enhanced biomass production, which was consistent with the results of yeast extracellular fluid supplementation. These findings conclusively demonstrate that acetate serves as the key mediating factor in the observed effects within the co-culture system.

 

Comments 8: Line 233 (Figure 3 legend): The p-values are mistakenly represented as ***P < 0.001, **P < 0.005, *P < 0.01, which does not follow standard statistical convention (commonly: *P<0.05, **P<0.01, ***P<0.001).

Response 8: Thank you for pointing out the inconsistency in the p-value annotation in Figure 3. We have corrected the legend to follow standard statistical conventions.

 

Comments 9: Figure 2 legend: Units for pigment content (mg/kg or mg/g wet biomass) are inconsistent or unclear. Consistent units across the manuscript are necessary for clarity.

Response 9: Thank you for the comment. We have unified all pigment content units to mg/kg in the text, figures, and legends for consistency.

 

Reviewer 2 Report

Comments and Suggestions for Authors

 

This study investigated the potential of co-culturing A. platensis with S. cerevisiae to enhance nutritional value and sensory quality. The co-culture led to increased levels of chlorophylls, carotenoids, and aroma compounds. In addition, acetic acid was found to upregulate genes involved in the biosynthesis of flavor precursors and to promote biomass accumulation.

 

 

The following suggestions are offered for revision.

1. In the co-culture of A. platensis and S. cerevisiae, chlorophyll and carotenoid levels increased; however, the color of the culture changed from dark blue-green to light green. This observation should be further discussed.

 

 

Line 88 (Figure 1C)

The resolution of Figure 1C is not enough.

The four images shown in Figure 1C require a description

 

Line 162~165

“In the present study, a total of 102 volatile compounds were identified in the monocultured and co-cultured microalgae using SPME-GC-MS, and they included 31 ketones, 15 alcohols, 15 aldehydes, 8 nitrogenous compounds, 5 phenols, 4 sulfur compounds, 3 organic acids, 2 furans, 2 esters, and 16 hydrocarbons (Table 1).”

Several numbers are incorrect. For example, there are 29 ketones, 13 alcohols, 17 aldehydes, and 1 ester. In addition, the total number of volatile compounds is also incorrect.

 

 

Line 258 (Table 2)

It should be 18S, not 16S.

 

Line 280 (Figure 4)

(1)Figure 4a should be described in the manuscript.

(2) Two boxes shown in Figure 4a require a description

 

Line 451, Table S1, S2

Trace metal

 

Author Response

Comments 1: In the co-culture of A. Platensis and S. cerevisiae, chlorophyll and carotenoid levels increased; however, the color of the culture changed from dark blue-green to light green. This observation should be further discussed.

Response 1: Thank you for your insightful comment regarding the color change in A. platensis during co-culture with S. cerevisiae. We appreciate your observation and have addressed it by discussing this observation as a potential dilution effect from yeast-derived biomass or altered pigment packaging in co-cultured microalgae (Lines 128-131).

The observed chromatic alteration, concomitant with increased chlorophyll a and carotenoid concentrations (Figure 2d), presumably represents a dilution phenomenon attributable to yeast-derived biomass accumulation or modified pigment organization in the co-cultured microalgae system.

 

Comments 2: Line 88 (Figure 1C) The resolution of Figure 1C is not enough. The four images shown in Figure 1C require a description

Response 2: Thank you for your comment regarding Figure 1C. We have carefully rechecked the original file and confirm that Figure 1C was submitted in high-resolution format. The apparent low resolution in the reviewed version may be due to file conversion during the submission process.

Thank you for your keen observation regarding the morphology of Arthrospira platensis (FACHB-902) in Figure 1. We have added a clarifying statement in the revised manuscript (see Line 92-93):

Microscopic morphology of A. platensis: (1) monoculture; (2) yeast/microalgae ratio 1:1000;

 

Comments 3:Line 162~165. “In the present study, a total of 102 volatile compounds were identified in the monocultured and co-cultured microalgae using SPME-GC-MS, and they included 31 ketones, 15 alcohols, 15 aldehydes, 8 nitrogenous compounds, 5 phenols, 4 sulfur compounds, 3 organic acids, 2 furans, 2 esters, and 16 hydrocarbons (Table 1).” Several numbers are incorrect. For example, there are 29 ketones, 13 alcohols, 17 aldehydes, and 1 ester. In addition, the total number of volatile compounds is also incorrect.

Response 3: Thank you for your careful review and for identifying the inaccuracies in the volatile compound numbers reported in Lines 170–175.

a total of 98 volatile compounds were identified in the monocultured and co-cultured microalgae using SPME-GC-MS, and they included 29 ketones, 17 aldehydes, 13 alcohols, 8 nitrogenous compounds, 5 phenols, 4 sulfur compounds, 3 organic acids, 2 furans, 1 esters, and 16 hydrocarbons (Table 1).

 

Comments 4: Line 258 (Table 2). It should be 18S, not 16S.

Response 4: Thank you for the comment. We confirm Arthrospira platensis is a prokaryotic cyanobacterium, and the correct ribosomal gene is 16S (not 18S).

 

Comments 5: Line 280 (Figure 4) (1) Figure 4a should be described in the manuscript. (2) Two boxes shown in Figure 4a require a description

Response 5: Thank you for your comments. We have now described Figure 4a in the manuscript (Lines 260–264) and clarified that the two boxes in Figure 4a represent volatile flavor compounds with floral and fruity aromas derived from precursor cleavage (Lines 289–291).

Figure 4a specifically illustrates the biosynthetic pathways of flavor precursors (β-carotene, linoleic acid, and γ-linolenic acid), including the fatty acid biosynthesis pathway and the methylerythritol phosphate pathway (MEP pathway). The subsequent cleavage of these precursors generates volatile flavor compounds with floral and fruity aromas (indicated by boxes in the diagram).

Boxes represent volatile flavor compounds (with floral and fruity notes) generated through the cleavage of β-carotene, linoleic acid, and γ-linolenic acid.

 

Comments 6: Line 451, Table S1, S2. Trace metal

Response 6: Thank you for your comment. We have revised the manuscript to replace this term with the more standard and widely used expressions “trace elements”.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors develop a research to solve a technological problem of spirulina with a very interesting bioprocess, by the use of co-cultures with S. cerevisiae they increase the nutrient value and improve the sensory quality, they also prove by transcriptome and quantitative PRC analysis, the roll of acetic acid produced by S. cerevisiae on the metabolism of spirulina, to explain its effect on the PUFA and B-carotene synthesis. Methodology is clear, as well as the way the results are presented and discussed. I did not find mistakes in the text, I think it has the quality to be published in the present form.

Author Response

Thank you very much for your positive evaluation of our study and your insightful comments. We truly appreciate your recognition of the novelty of using A. platensis and S. cerevisiae co-culture to enhance nutritional and sensory qualities, as well as your emphasis on the key findings regarding the role of acetic acid in regulating spirulina metabolism (particularly PUFA and β-carotene synthesis) through transcriptomic and qPCR analyses.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have answered to all my comments.

Comments on the Quality of English Language

Minor revision of English along the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors response well, so I have no more suggestion.