Low-Cost Production Process of Saccharomyces cerevisiae Yeast for Craft Beer Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall Impression

This study is systematically designed, utilizing a combination of Plackett-Burman and full factorial designs for medium optimization, and successfully validates the process at both laboratory and pilot scales. The data provided is generally supportive. The core innovation lies in utilizing brewery by-products to develop a low-cost medium and demonstrating its performance can surpass that of a synthetic medium. This holds significant value for the craft beer industry in resource-limited regions.

However, the manuscript exhibits some deficiencies in scientific rigor, data presentation, depth of results interpretation, and language clarity, necessitating major revisions.

 

Specific Revision Suggestions

1.The abstract lacks key quantitative results (e.g., specific cost reduction percentage) and statements of statistical significance. It is suggested to incorporate specific numerical values and statistical significance for key results and explicitly mention cost estimates or the percentage reduction achieved.

2. The description of the research gap in introduction is not sufficiently critical and fails to clearly distinguish this work from previous studies. For instance, while cost is mentioned, the limitations of existing low-cost media are not systematically reviewed. Please strengthen the end of the introduction to clearly state the specific scientific hypothesis and the precise technical bottleneck this study aims to address. Add a paragraph briefly reviewing other existing low-cost alternative media for yeast propagation (e.g., molasses, other agricultural wastes) and their advantages/disadvantages, thereby highlighting the uniqueness and innovation of the substrates used in this study.
3. The final chemical composition (e.g., sugar concentration, amino nitrogen content) of the malt and yeast extracts in the alternative medium is not quantified, making the medium composition unclear and difficult to replicate. Please supplement the Methods with chemical analysis data for key components of the alternative medium (e.g., sugar profile via HPLC, nitrogen content via Kjeldahl method).
4. In the statistical methods section, specify the exact software, packages, versions used, and describe the ANOVA post-hoc tests (e.g., Tukey HSD) and model validation procedures.
5. In Figure 1, error bars (SD/SEM) are absent, preventing assessment of variability between replicates. Please add error bars to all bar graphs and growth curves, stating the number of replicates (n=?).
6. A direct side-by-side comparison of the low-cost and synthetic media growth kinetics and final yield at the same cultivation scale is missing, weakening the argument. It is suggested to include a combined figure directly comparing growth kinetics and final yield of the synthetic and low-cost media at the same laboratory scale.
7. The significant difference between the two pilot-scale validation results is merely mentioned but not analyzed for potential causes. Please discuss potential reasons for the discrepancy between the two pilot-scale runs in the text, such as inoculum consistency, mass transfer differences, or batch-to-batch variation in the substrates.
8. Excessively repeats results without delving into underlying mechanisms. Why does the low-cost medium perform better? Does it provide richer growth factors, micronutrients, or is there a synergistic effect? It is suggested to discuss the potential physiological and metabolic mechanisms behind the superior performance of the low-cost medium. For example, spent yeast extract might contain glutathione, vitamins, or other factors absent in commercial extracts that promote growth.
9. Fails to quantitatively compare key performance indicators (e.g., growth rate, biomass yield) with those reported in similar studies using other low-cost substrates. Please add a table systematically comparing key parameters from this study (max biomass, growth rate) with those from literature using other low-cost substrates (e.g., molasses, whey permeate).
10. Mentions variability in by-product composition only briefly, without addressing it as a core challenge. It is better to strengthen the limitations analysis, and based on this, propose concrete future research directions for monitoring and standardizing the quality of the by-products.
11. The conclusions are overly general, resembling a restatement of the abstract, and fail to distill the core contributions and forward-looking perspectives of the study. Please rewrite the conclusions: start with 1-2 sentences summarizing the most fundamental finding, clearly articulate the practical application potential and expected impact of this technology on the industry, and propose specific future research directions, e.g., conducting pilot-scale fermentations, evaluating the metabolic performance and flavor impact of the yeast produced in actual beer fermentation.
12. It is recommended to seek professional English editing services or assistance from a native English speaker for a thorough revision.
13. Formatting: ensure all figure and table titles/captions are complete and self-explanatory, standardize the reference format, check and ensure all in-text citations correctly correspond to entries in the reference list.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I do not reccomend this paper for publication. My detailed review is provided in the attached file.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript illustrates an interesting and effective way to propagate yeasts for beer production using brewery by-products as a nitrogen source, with an approach in line with a sustainable point of view. The work is well designed and validated for an optimized synthetic culture medium, tested at laboratory scales, and a low-cost culture medium tested at both laboratory and pilot scale. Only minor revisions are needed to improve the interpretation of tables, and a few sentences need to be checked, as specified below. The English is fine, with only a few errors to correct, and references are appropriate.

Only a few minor issues need to be addressed before publication:

Line 46: please add “dioxide” after “carbon”

Line 121: Please check the sentence

Lines 147-148 It appears that the sentence refers to the content of table 1 (not S2 as reported in the text), please check.

Caption Fig S2: Please check last sentence

Lines 215-216: I could not find the results obtained from conventional or commercial synthetic media, could you clarify how was the comparison carried out? If it wasn’t, please delete or change the sentences accordingly.

Lines 244 and 251: please add references to Table S3, to help identify the various treatments

Lines 260-261: Please check the sentence

Table S4: Table S4 needs reformatting to show more clearly to which medium and scale the results refer to. From this table, it seems that laboratory scale experiments refer only to Optimized synthetic culture medium, and that Low-cost culture medium experiments were only performed in 83L batches, but this does not overlap with what is described in the text.

Line 312: in the second validation the doubling time is not shorter than in the laboratory scale, and the differences between laboratory and pilot scales do not appear to be significant: please delete “even shorter:”.

Line 349: please doublecheck ref. 28

Lines 404 and 427: see comment to lines 215-216

Comments on the Quality of English Language

The overall quality of English language  is fine, but in a few sentences it looks as if some words are gone missing; please check throughout.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript analyzes the growth of brewing yeasts on various low-value media. The work has great biotechnological potential, but I have a number of comments.

Abstract

18 propagation methods ??? Perhaps it would be more appropriate to use the term growth, cultivation, accumulation of biomass.

L 98 Culture conditions included an incubation at 25°C for 48 hours. – The temperature for yeast cultivation is traditionally 28-30 degrees.

L 130 the authors used zinc sulfate heptahydrate (LOBA Chemie, India) as a zinc source. It's unclear why the authors used a separate zinc source, given that yeast extract typically contains all the essential micronutrients for growth. If this is required by specific growth conditions, this should be clarified.

It's unclear why the authors used such a labor-intensive cell counting method. Wouldn't it have been simpler to present data based on optical density at 600 nm? Viability could have been reliably determined using flow cytometry.

Table 1, 2 – What is meant by Agitation? Mixing? Aeration?

2.7. Kinetic Analysis – The parameters specified in the equations should be deciphered.

Results

The authors indicate that the key factors for optimal growth were the concentration of the substrate, nitrogen source, and zinc, as well as mixing, which is obvious. For clarity, the specific medium and cultivation parameters used should be specified.

Figure 1. Treatment options should be explained in the figure caption. Statistical data should also be provided.

Figure 2. In my opinion, 8 points on the curve are insufficient to plot growth kinetics over 70 hours. There are also no statistics for these points.

Figures 3, 4 – no statistics. In Figure 4, it is noteworthy that despite radically different growth kinetics for yeast cell biomass (A, C), the logarithmic dependence is very similar. Is this an error?

It is also unclear whether specific supplements were added during growth scaling in the bioreactor. If so, this should be noted.

Discussion

In this section, the rationale for using zinc as an essential medium component should be provided. What about other micronutrients? What is their significance? This section also contains repetitions that should be eliminated, focusing on the specific results obtained by the authors.

The reference list is not formatted according to the journal's guidelines.

Comments on the Quality of English Language

Moderate editing

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has basically answered all the questions I raised, and I have no more suggestions.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I have familiarized myself with the revised version of this manuscript as well as Authors response. In my opinion, extensive clarifications provided in the text along with clarifications in Authors response have dispeled all my concerns I've had with the origial version of this manuscript. Extensive explanaitions provided in revised version allows now to track the general idea that the Authors have had from the very beginning and provided scientifically and practically sound document. Although I still do not feel that in general this paper is a complete novelty I do not have a problem with that now while Authors have explicitly placed their idea and applicability within Ecuador and surroundings area with locally provisioned feedstocks and equipment. This paper might be an 'adevrtisement' for the Authors bioprocess development center that will provide pure yeast strains for local applications. I now reccomend this paper for publication.

Author Response

We sincerely thank Reviewer 2 for the careful re-evaluation of our revised manuscript and for the positive recommendation for publication. We truly appreciate the acknowledgment of our clarifications and the recognition that the revised version now presents a scientifically sound and contextually relevant contribution.
We are also grateful for the reviewer’s understanding of the applied nature of our work and its significance for local bioprocess development and sustainable yeast production in Ecuador and the Andean region. Thank you again for your thoughtful feedback throughout the review process, which has greatly helped us improve the overall quality and clarity of the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Overall, I am satisfied with the work the authors have done to improve the manuscript.

However, I must leave a few questions unanswered.

On lines 258 and 262, u is used instead of µ. This should be corrected.

In Table S2, the p values for potassium phosphate and stirring exceed 0.05, indicating that they are unreliable.

I also disagree with the authors' responses to the comment regarding growth dynamics. I believe that eight data points are insufficient for calculating kinetic parameters. For example, in Figure 3A, there is a clear jump in biomass accumulation at ~70 hours of growth, which the authors do not explain. What could be the cause? Additional data points, for example, at 75 and 80 hours of growth, could provide an answer to this question. The same can be said about Figure 4A, which lacks data points at 60 and 65 hours. Perhaps growth fluctuations occurred during this period.

The role of zinc is not entirely clear. The authors point to its significant role in cellular processes, ignoring, for example, other microelements—magnesium, manganese, copper, and iron. If preliminary experiments to optimize the environment were conducted, these microelements should have been tested as well.

Otherwise, I believe the article can be published taking these comments into account.

Comments on the Quality of English Language

Moderate editing

Author Response

Please see the attachment.

Author Response File: Author Response.pdf