Review Reports - Biomass Production of <i>Chlorella vulgaris</i> var. <i>vulgaris</i> TISTR 8261 During Cultivation in Modified Food Industry Wastewater

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper describes the use of industrial wastewater of different sources for the enhancement of the algal biomass. The paper has merits and is publishable but after addressing the following:

1- Thorough analysis of wastewater content which include the identification of different organic or inorganic compounds that are found in the wastewater. This will certainly affect algal growth

2- What was the count of cells in the initial inoculum? or the starting biomass in all treatments?

3- When evaluating, growth, did you take samples from the growing cultures to measure spectrophometrically? wouldn't that affect volume and concentration of cells? How did you perform the growth experiment in details

4- I would recommend using Cannoical principal compoenet analysis

4- The specific concentrations of supplements 17.4 mM so-dium acetate significantly improved algal biomass yield. Further optimization with 3.7 mM NH₄Cl, 1.0 mM KH₂PO₄, 0.2 mM MgSO₄, and

How did you come up with these specific concentrations

5- Autoclaving with the autoclave , wouldn't that be destructive to some of the components present in wastewater, wouldn't it be better to sterilize by a membrane filtration using filtration unit

6- What are the indications gained from measuring BOD and COD. Please add explanation for measuring those parameters and their importance . What about their values after autoclaving ?

Author Response

Reviewer’s Comment 1:

Thorough analysis of wastewater content which include the identification of different organic or inorganic compounds that are found in the wastewater. This will certainly affect algal growth.

Response:

We thank the reviewer for this valuable suggestion. In the present study, we focused on measuring COD and BOD as representative parameters of the organic load in wastewater, since these indicators provide relevant information about the overall organic matter available to support algal growth. However, we did not perform detailed identification of specific organic or inorganic compounds. We agree that such analyses would provide further insights, and this point will be considered in our future work.

Reviewer’s Comment 2:

What was the count of cells in the initial inoculum? or the starting biomass in all treatments?

Response:

We thank the reviewer for this comment. The initial cell concentration in all treatments was adjusted to an optical density at 750 nm (OD₇₅₀) of approximately 0.1, which corresponds to 7.5 × 10⁴ cells mL^-1. This information has been added in the Materials and Methods section (Section 2.3, highlighted in green) of the revised manuscript.

Reviewer’s Comment 3:

When evaluating, growth, did you take samples from the growing cultures to measure spectrophometrically? wouldn't that affect volume and concentration of cells? How did you perform the growth experiment in details?

Response:

We thank the reviewer for this comment. The growth of C. vulgaris var. vulgaris TISTR 8261 was monitored by measuring the optical density at 750 nm (OD₇₅₀) using a UV-visible spectrophotometer. For each measurement, a small aliquot of 1 mL was taken from the culture. The removal of this small volume did not significantly affect the overall culture volume (100 mL per flask) or cell concentration. This explanation and the specific sample volume have now been added to the revised manuscript in Materials and Methods section (Section 2.4, highlighted in green) to clarify the procedure.

Reviewer’s Comment 4:

I would recommend using Cannoical principal compoenet analysis.

Response:

We appreciate the reviewer’s suggestion regarding the use of Canonical Principal Component Analysis (CPCA). However, in our study, we investigated the effect of one factor at a time on algal growth in wastewater. Since each experiment was designed to assess the response to a single variable independently, one-way ANOVA is appropriate and sufficient for analyzing the differences between treatments. Therefore, we believe that the current statistical approach adequately addresses the experimental design and the objectives of our study.

Reviewer’s Comment 5:

The specific concentrations of supplements 17.4 mM so-dium acetate significantly improved algal biomass yield. Further optimization with 3.7 mM NH₄Cl, 1.0 mM KH₂PO₄, 0.2 mM MgSO₄, and How did you come up with these specific concentrations?

Response:

We thank the reviewer for this comment. The specific concentrations of NH₄Cl (3.73 mM), KH₂PO₄ (1.0 mM), and MgSO₄ (0.2 mM) used in our experiments were chosen to match those in the standard TAP (Tris-Acetate-Phosphate) medium, which is widely applied for Chlorella vulgaris cultivation. This ensured that the cultures received appropriate nutrient levels to support optimal algal growth. The revised manuscript now includes this clarification in the Materials and Methods section (Section 2.6 highlighted in green).

Reviewer’s Comment 6:

Autoclaving with the autoclave, wouldn't that be destructive to some of the components present in wastewater, wouldn't it be better to sterilize by a membrane filtration using filtration unit.

Response:

We appreciate the reviewer’s comment. In our study, autoclaving at 121 °C for 15 minutes was used to ensure that no contaminating microorganisms were present during the experiments. While we acknowledge that autoclaving may affect some components in the wastewater, preventing microbial contamination was essential for the controlled assessment of algal growth. This point has been clarified in Section 2.2 (highlighted in green).

Reviewer Comment 7:

What are the indications gained from measuring BOD and COD. Please add explanation for measuring those parameters and their importance . What about their values after autoclaving ?

Response:

We appreciate the reviewer’s valuable suggestion. In response, we have added clarification in the Discussion (Section 4.2, highlighted in green) to explain the importance of BOD and COD measurements as indicators of the organic load in raw effluents. Furthermore, we have addressed the reviewer’s concern regarding post-autoclaving values by noting that, while autoclaving may slightly reduce BOD and COD values (Sahoo, 2017), such reductions are generally minor and unlikely to significantly influence algal growth in our cultivation experiments.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a well-designed, timely, and highly relevant study that addresses the dual challenges of industrial wastewater management and sustainable microalgal biomass production. The research is methodologically robust, following a logical progression from wastewater screening and characterization to systematic medium optimization and a successful scale-up demonstration. The results are significant and impactful, particularly the achievement of a higher biomass yield (8.436 g L⁻¹) in the optimized wastewater medium compared to the standard laboratory TAP medium. The narrative is clear, the data is presented effectively, and the work represents a valuable contribution to the fields of applied phycology, biotechnology, and environmental science. The authors are commended for their thorough approach and the clarity of the manuscript.

However, there are several critical issues related to methodology, data interpretation, and contextualization that must be addressed to ensure the scientific rigor and overall impact of the publication. The required revisions are substantial but do not necessitate new experimental work; they focus on re-analysis of existing data, re-framing of key interpretations in the Discussion, and acknowledging methodological limitations.

Major Revisions (Essential for Acceptance)

The following major points must be addressed before the manuscript can be considered for publication:

This is The most urgent issue is critical flaw in chlorophyll determination (Section 2.5). There is an inconsistency between the stated measurement wavelengths (665 nm and 650 nm) and those used in the equation (A₆₆₄ and A₆₄₇). More importantly, the provided formula for chlorophyll in 90% methanol is non-standard and its source is not cited. This calls into question the accuracy of all reported chlorophyll data.

The authors must verify the source and validity of their equation. It is strongly recommended that they recalculate all chlorophyll concentrations using a well-established, standard, and properly cited equation for Chlorella in 90% methanol (e.g., from Ritchie, 2006 or Porra et al., 1989). All figures and text referencing chlorophyll content must be updated accordingly.

The study achieved an outstanding biomass yield under a very low light intensity (30 µmol photons m⁻² s⁻¹). At such a high cell density, the culture would be severely light-limited due to self-shading. The Discussion currently fails to acknowledge that the high productivity was almost certainly driven by the mixotrophic metabolism of sodium acetate, not by photosynthesis. The Discussion must be revised to include a thorough analysis of this interplay. The authors should explicitly state that light became a limiting factor at high density and that the culture’s success demonstrates the power of mixotrophy in this system.
The wastewater was sterilized via autoclaving, a process known to alter the chemical composition of complex media by causing precipitation of minerals (e.g., phosphates, iron) and affecting organic compounds. This means the algae were grown in a medium chemically different from what was initially characterized. The authors must add a paragraph to the Discussion acknowledging this methodological limitation. They should discuss the potential effects of autoclaving on nutrient bioavailability and how this might have influenced the results. Sterile filtration should be mentioned as a preferred alternative for future studies.
The finding that the optimized PFPP biomass had a significantly higher ash content (11.7%) than TAP-grown biomass (3.5%) is a critical result with major practical implications. The current discussion is good but incomplete. The Discussion should be expanded to (a) explicitly attribute the high ash to the mineral load from both the base wastewater and the nutrient supplements, and (b) more deeply explore the consequences, clearly stating that while beneficial for biofertilizer or feed fortification, it reduces the biomass quality for biofuel applications and may impact digestibility in feed.

Minor Revisions

Line numbers are missing.
In the Discussion, explicitly link the superior growth in PFPP (Section 3.2) to its higher initial concentrations of nitrogen and phosphorus compared to the other wastewaters (Table 1).

Expand the discussion on why the 0.2X trace mineral concentration was optimal and the 1.0X concentration was inhibitory, proposing that the baseline wastewater provided enough of certain metals to make the 1.0X addition toxic.
Strengthen the Conclusion by more explicitly mentioning the high nutrient removal rates achieved, tying back directly to the bioremediation goal.
Tailor the suggested applications in the Conclusion to the actual biomass profile (high protein/mineral, low lipid), emphasizing its suitability for animal feed and biofertilizer rather than general "bio-based industries."
Add a brief, forward-looking sentence to the Conclusion suggesting next steps (e.g., pilot-scale trials, outdoor cultivation).
Fix all typographical errors noted in the section-by-section review (e.g., "Curde," "Maccronutrients,") and verify the section numbering.
Ensure consistent and precise use of terms like "photobioreactor" vs. "culture vessel." Clarify the likely typo of a "100-gram sample" for nutritional analysis.

Author Response

Major Revisions (Essential for Acceptance)

The following major points must be addressed before the manuscript can be considered for publication:

This is The most urgent issue is critical flaw in chlorophyll determination (Section 2.5). There is an inconsistency between the stated measurement wavelengths (665 nm and 650 nm) and those used in the equation (A₆₆₄ and A₆₄₇). More importantly, the provided formula for chlorophyll in 90% methanol is non-standard and its source is not cited. This calls into question the accuracy of all reported chlorophyll data.

Response:
We sincerely thank the reviewer for carefully identifying this important issue. We acknowledge that there was an inconsistency in Section 2.5 between the stated measurement wavelengths and the equation presented. We would like to clarify that this was a typographical error in the manuscript preparation. In fact, all chlorophyll concentrations were calculated using the correct equation described by Lee and Shen (2004) with absorbance values at 665 nm and 650 nm. We have now corrected the text and equation in Section 2.5 accordingly (highlight in yellow). Importantly, the chlorophyll data reported in the results and discussion were all originally calculated using this correct formula, so no recalculation of the data was required. We sincerely apologize for the oversight in reporting the equation and thank the reviewer for drawing our attention to this error.

The study achieved an outstanding biomass yield under a very low light intensity (30 µmol photons m⁻² s⁻¹). At such a high cell density, the culture would be severely light-limited due to self-shading. The Discussion currently fails to acknowledge that the high productivity was almost certainly driven by the mixotrophic metabolism of sodium acetate, not by photosynthesis. The Discussion must be revised to include a thorough analysis of this interplay. The authors should explicitly state that light became a limiting factor at high density and that the culture’s success demonstrates the power of mixotrophy in this system.

Response:
We thank the reviewer for this insightful comment. We apologize for the confusion caused by a typographical error in the Materials and Methods section. In the flask experiments, cultures were grown at 30 µmol photons m⁻² s⁻¹, but in the 3.5 L culture bottles the actual light intensity was 100 µmol photons m⁻² s⁻¹, not 30 µmol photons m⁻² s⁻¹ as incorrectly stated in the original text. This has now been corrected in the revised manuscript (Section 2.3, highlight in yellow).

Nevertheless, we fully agree with the reviewer that at high cell density, self-shading inevitably leads to light limitation, and that mixotrophic metabolism from sodium acetate significantly contributed to the observed high productivity. We have revised the Discussion to explicitly acknowledge this interplay, emphasizing that while photosynthesis was limited under dense culture conditions, the success of the system demonstrates the strong potential of mixotrophy in sustaining algal growth and biomass yield (Section 4.5, highlight in yellow).

The wastewater was sterilized via autoclaving, a process known to alter the chemical composition of complex media by causing precipitation of minerals (e.g., phosphates, iron) and affecting organic compounds. This means the algae were grown in a medium chemically different from what was initially characterized. The authors must add a paragraph to the Discussion acknowledging this methodological limitation. They should discuss the potential effects of autoclaving on nutrient bioavailability and how this might have influenced the results. Sterile filtration should be mentioned as a preferred alternative for future studies.

Response:
We thank the reviewer for highlighting this important methodological issue. We agree that autoclaving complex wastewater media can alter its chemical composition, particularly by causing precipitation of phosphate and iron salts and modifying certain organic compounds. As a result, the actual nutrient environment experienced by the algae may have differed from the initial chemical characterization. We have now added a paragraph in the Discussion to acknowledge this limitation. Specifically, we note that autoclaving may have reduced the bioavailability of some nutrients and thereby influenced algal growth performance. For future studies, sterile filtration will be considered as a preferable sterilization method to better preserve the native chemical profile of wastewater while ensuring microbial safety (Section 4.5, highlight in yellow).

The finding that the optimized PFPP biomass had a significantly higher ash content (11.7%) than TAP-grown biomass (3.5%) is a critical result with major practical implications. The current discussion is good but incomplete. The Discussion should be expanded to (a) explicitly attribute the high ash to the mineral load from both the base wastewater and the nutrient supplements, and (b) more deeply explore the consequences, clearly stating that while beneficial for biofertilizer or feed fortification, it reduces the biomass quality for biofuel applications and may impact digestibility in feed.

Response:

We thank the reviewer for highlighting this important point. In response, we have expanded the Discussion section (Section 4.6, highlighted in yellow) to explicitly attribute the elevated ash content in PFPP-grown biomass to the combined mineral load originating from the wastewater base medium and the added nutrient supplements. Furthermore, we have deepened the discussion of the practical consequences: while the high ash fraction can be advantageous for applications such as biofertilizer production or animal feed fortification (due to its enrichment in essential minerals), it simultaneously reduces the suitability of the biomass for biofuel production by lowering lipid quality, complicating transesterification, and promoting fouling during thermochemical conversion. We also note that excessive mineral content may impair digestibility if used as feed without appropriate balancing. These revisions directly address the reviewer’s concerns by linking the observed result to its causes and clarifying the implications for different end-use applications.

Minor Revisions

Line numbers are missing.
In the Discussion, explicitly link the superior growth in PFPP (Section 3.2) to its higher initial concentrations of nitrogen and phosphorus compared to the other wastewaters (Table 1).

Response:

We thank the reviewer for this suggestion. As highlighted in the revised manuscript (Section 4.2, highlighted in yellow), the superior growth observed in PFPP-grown cultures has been explicitly linked to its higher initial concentrations of total nitrogen, total phosphorus, and sulfate, which are critical macronutrients supporting cell growth and division.

Expand the discussion on why the 0.2X trace mineral concentration was optimal and the 1.0X concentration was inhibitory, proposing that the baseline wastewater provided enough of certain metals to make the 1.0X addition toxic.

Response:

We thank the reviewer for this insightful comment. In the revised manuscript (Section 4.4, highlighted in yellow), we have expanded the discussion to explain why the 0.2X trace mineral concentration was optimal, whereas the 1.0X concentration was inhibitory. Specifically, we clarify that the baseline PFPP wastewater already contained sufficient levels of essential trace metals, so only minimal supplementation was needed to support robust algal growth. In contrast, the higher 1.0X concentration likely caused an excess of certain metals, leading to potential toxicity or metabolic burden, such as oxidative stress or inhibition of enzymatic functions, which resulted in reduced growth. This revision explicitly links the observed dose-dependent growth response to both the baseline mineral content of the wastewater and the supplemented trace minerals, addressing the reviewer’s concern.

Strengthen the Conclusion by more explicitly mentioning the high nutrient removal rates achieved, tying back directly to the bioremediation goal.

Response :

We thank the reviewer for this valuable suggestion. In the revised manuscript, we have strengthened the Conclusion (Section 5, highlighted in yellow) by explicitly highlighting the high nutrient removal rates achieved in PFPP-grown C. vulgaris var. vulgaris TISTR 8261 cultures.

Tailor the suggested applications in the Conclusion to the actual biomass profile (high protein/mineral, low lipid), emphasizing its suitability for animal feed and biofertilizer rather than general "bio-based industries."

Response:
We thank the reviewer for this valuable suggestion. In the revised manuscript, we have updated the Conclusion (Section 5, highlighted in yellow) to tailor the suggested applications to the actual biomass profile, which is high in protein and minerals but relatively low in lipids.

Add a brief, forward-looking sentence to the Conclusion suggesting next steps (e.g., pilot-scale trials, outdoor cultivation).

Response:
We thank the reviewer for this suggestion. A brief, forward-looking statement has been added to the Conclusion (Section 5, highlighted in yellow) to suggest next steps.

Fix all typographical errors noted in the section-by-section review (e.g., "Curde," "Maccronutrients,") and verify the section numbering.

Response:
We thank the reviewer for pointing out the typographical errors and section numbering issues. All identified typographical mistakes (e.g., “Curde,” “Maccronutrients”) have been corrected, and the section numbering throughout the manuscript has been carefully verified and updated where necessary to ensure consistency and accuracy.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The content of the manuscript concerns important issues in the field of circular economy.

The authors submitted a paper on the optimization of the cultivation of the microalgae Chlorella vulgaris var. vulgaris TISTR 8261 using food industry wastewater supplemented with sodium acetate, which significantly increased the biomass of Chlorella vulgaris var. vulgaris TISTR 8261.

The algae cultivation conditions were optimized by supplementing them with sodium acetate and macro and micronutrients.

The possibility of using industrial wastewater in a circular economy and effective wastewater bioremediation was demonstrated.

They demonstrated the ability of C. vulgaris to effectively absorb N, P, and S compounds and produce biomass.

The text provides valuable information on culturing Chlorella vulgaris var. vulgaris TISTR 8261 in industrial wastewater.

However, it has several weaknesses that the authors should address in the manuscript. A low-cost source of nutrients is indicated, but the cost-effectiveness of this technology is not demonstrated. The authors describe TAP medium as economically unviable for industrial applications – please justify this. The costs of culturing in PFPP wastewater should be compared with those in TAP medium.

The authors described algae production in 3.5-liter bottles but did not address the subsequent steps required to achieve large-scale production. This information needs to be supplemented. Potential negative effects of excessive ash content were noted. The authors should also note the positive effects of ash, for example, on agricultural crops, as described in: https://doi.org/10.1038/s41598-023-45733-9

https://doi.org/10.3390/cells12020289

The authors only identified a single period of wastewater sampling. They did not account for possible seasonal changes or differences in the composition of this wastewater.

Author Response

Reviewer’s comment 1:

Response:
We thank the reviewer for this valuable comment. We agree that cost-effectiveness is essential for evaluating the feasibility of large-scale algal cultivation. As suggested, we performed a direct cost comparison between TAP medium and Modified PFPP wastewater. The estimated cost of preparing TAP medium was 2.45 USD L⁻¹, whereas cultivation in Modified PFPP wastewater required only 0.12 USD L⁻¹, resulting in a ~20-fold reduction in cost. This demonstrates the significant economic advantage of PFPP wastewater utilization, in addition to its contribution to wastewater remediation. We have added this cost analysis and corresponding discussion in the revised manuscript (Section 4.5 highlighted in blue).

Reviewer’s comment 2:

The authors described algae production in 3.5-liter bottles but did not address the subsequent steps required to achieve large-scale production. This information needs to be supplemented.

Response:
We appreciate the reviewer’s comment. Our current study focused on laboratory-scale cultivation in 3.5 L bottles to evaluate the growth performance of the selected microalgae in PFPP wastewater. We agree that scaling up is an important consideration for future applications. Large-scale production would typically require cultivation in open raceway ponds or closed photobioreactors, where parameters such as light penetration, mixing, aeration, and contamination control play a crucial role. These aspects have been briefly conclusios in the revised manuscript (Section 5 highlighted in blue).

Reviewer’s comment 3:

Potential negative effects of excessive ash content were noted. The authors should also note the positive effects of ash, for example, on agricultural crops, as described in: https://doi.org/10.1038/s41598-023-45733-9 https://doi.org/10.3390/cells12020289

Response:

We thank the reviewer for this valuable suggestion. We agree that, in addition to potential negative effects of high ash content, positive effects should also be noted. In the revised manuscript, we have added a discussion highlighting that mineral-rich microalgal ash can improve soil quality and enhance crop performance by contributing essential nutrients and bioactive compounds (Section 4.6 highlighted in blue). References suggested by the reviewer have been included to support this point (doi:10.1038/s41598-023-45733-9; doi:10.3390/cells12020289).

Reviewer’s Comment 4:

The authors only identified a single period of wastewater sampling. They did not account for possible seasonal changes or differences in the composition of this wastewater.

Response:

We thank the reviewer for this valuable comment. We acknowledge that wastewater composition can vary depending on seasonal changes, raw material inputs, and industrial processes. To minimize variability and ensure consistency across all experiments, wastewater was collected during a single sampling period. We have clarified this in the revised manuscript (Section 4.1, highlighted in blue).

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Phycology-3854937

The title needs to be corrected, as an example: Biomass production of Chlorella vulgaris var. vulgaris TISTR 8261 during cultivation in modified food industry wastewater.
First sentence of the abstract: the word «bioremediation» should be removed.
«Chlorophyll content» in the text should be replaced with «chlorophyll concentration», and authors should also provide data on the content (% of dry weight).
Complete the keywords with the object of study, cultivation mode, wastewater clarification, etc.
Update: «They can grow under mixotrophic or heterotrophic conditions by utilizing organic carbon compounds present in wastewater [5-6]».
Make a generalization on the biochemical composition based on the analysis of several studies: «Among these, Chlorella vulgaris is recognized as one of the most promising species due to its rapid growth, resilience to diverse environments, and high-value biochemical composition, including protein (~56.8%), lipids (~16.9%), carbohydrates (~5.9%), and pigments such as carotenoids and chlorophylls (1–2% DW) [16]».
Use the correct name of the medium (Bold’s basal medium).
It is necessary to expand the list of works in the direction of modifying the composition of nutrient media with the aim of increasing the productivity of biomass and target products: Effect of nutrient supply status on biomass composition of eukaryotic green microalgae; Influence of Nutrient Medium Composition on the Redistribution of Valuable Metabolites in the Freshwater Green Alga Tetradesmus obliquus (Chlorophyta) Under Photoautotrophic Growth Conditions, and others.
Clarification needed on products: «The PFPP facility produces ready-to-eat meals, frozen foods, and chilled foods».
Sections 2.1 and 2.2 contain repetitions.
The use of autoclaving will increase the cost of the proposed biotechnology.
The use of distilled water is not recommended; it is better to replace it with phosphate buffer: «Cells were harvested by centrifugation at 7,000 × g, 20 °C for 10 minutes, washed twice with sterile distilled water, and resuspended in wastewater».
Section 2.3 should be carefully edited to avoid confusion. What was the volume of inoculum (100 mL)?
Plus, it is necessary to provide a rationale for choosing such a low PPFD, and provide a description of aeration, inorganic carbon supply, pH changing... the use of the applied format, 3.5-L culture bottles (shading of cells) and a fairly low PPFD level does not allow to obtain good results in terms of culture productivity (which is also evident from the level of OD and chlorophyll in the cells).
Clarify: «Under cultivation in photobioreactor, C. vulgaris var. vulgaris TISTR 8261 was cultivated in 3.5-L culture bottles under controlled laboratory conditions».
Section 2.5: total chlorophyll?
The authors should explicitly state that they verified ANOVA assumptions (Shapiro–Wilk for normality and Levene’s for homogeneity, p > 0.05) before analysis, or else indicate the use of a nonparametric alternative (e.g., Kruskal–Wallis).
What were the levels of ammonium nitrogen in the wastewater initially, before modification?
«2.7. Nutritional Analysis of C. vulgaris var. vulgaris TISTR 8261» change to «2.7. Biomass characteristics of C. vulgaris var. vulgaris TISTR 8261»
Include in Table 1 the TAP composition (mg L^-1) to compare key components with their levels in alternative media.
Figure 4 should be replaced or supplemented to provide a complete picture of the cultivation so that readers can see how the culture was cultivated (provision of stirring, lighting, aeration...).
Use “unmodified PFPP” instead of “normal PFPP” and “modified PFPP” instead of “optimal PFPP”.
Section 2.4 Provide the equation for biomass productivity calculation and explain which timepoints were used for SGR and BP calculation (during the exponential phase of growth?).
Based on the growth indicators, I strongly ask the authors to review the data on dry weight; it is clear that Maximum Biomass yield (g L^-1) and Maximum Biomass production (g L^-1 day^-1) are calculated incorrectly and raise serious doubts (even based on the intensity of the culture medium in Figure 4).
Mixotrophic cultivation (with organic carbon sources) supply is creating a very high risk of contamination of the microalgae culture and obtaining inaccurate results. Please provide data about bacterial loading at the end of the cultivation period.

In addition, the authors should conduct separate experiments on wastewater treatment without the participation of algae in order to take into account or exclude the influence of accompanying microflora on remediation indicators, the weight of microbial biomass, and its indicators.

In general, the study should be supplemented with additional experiments, and the authors should adjust their calculations for growth indicators. The text is very difficult to read in some places, and the authors need to work on it more.

Author Response

Reviewer’s Comment 1:

The title needs to be corrected, as an example: Biomass production of Chlorella vulgaris var. vulgaris TISTR 8261 during cultivation in modified food industry wastewater.

Response:
We appreciate the reviewer’s helpful suggestion. Accordingly, the manuscript title has been revised to improve clarity and conciseness as follows: “Biomass production of Chlorella vulgaris var. vulgaris TISTR 8261 in modified food industry wastewater.” (Highlight in gray)

Reviewer’s Comment 2:

First sentence of the abstract: the word «bioremediation» should be removed.

Response:
We thank the reviewer for this suggestion. The word “bioremediation” has been removed from the first sentence of the Abstract to maintain focus on biomass production. The revised sentence now reads: “Industrial wastewater can serve as a low-cost nutritional source for sustainable microalgal biomass production.”

Reviewer’s Comment 3:

«Chlorophyll content» in the text should be replaced with «chlorophyll concentration», and authors should also provide data on the content (% of dry weight).

Response:
We appreciate the reviewer’s insightful comment. In the revised manuscript, the term “chlorophyll content” has been replaced with “chlorophyll concentration” throughout the text (Highlight in gray).

Reviewer’s Comment 4:

Complete the keywords with the object of study, cultivation mode, wastewater clarification, etc.

Response:
Thank you for the valuable comment. The keywords have been revised to incorporate the object of study, cultivation mode, and wastewater clarification, while keeping them concise. The revised keywords are:“ Chlorella; nutrient modulation; food industry wastewater; biomass production; culture bottle” (Highlight in gray).

Reviewer’s Comment 5:

Update: «They can grow under mixotrophic or heterotrophic conditions by utilizing organic carbon compounds present in wastewater [5–6]».

Response:
We thank the reviewer for this helpful suggestion. In accordance with the comment, we have revised the statement in the Introduction (highlighted in grey) to clarify that microalgae can grow under mixotrophic or heterotrophic conditions by utilizing organic carbon compounds present in wastewater. Furthermore, the reference list has been updated to include more recent literature (now cited as [5–7]).

Reviewer’s Comment 6:

Make a generalization on the biochemical composition based on the analysis of several studies: «Among these, Chlorella vulgaris is recognized as one of the most promising species due to its rapid growth, resilience to diverse environments, and high-value biochemical composition, including protein (~56.8%), lipids (~16.9%), carbohydrates (~5.9%), and pigments such as carotenoids and chlorophylls (1–2% DW) [16]».

Response:
We appreciate the reviewer’s suggestion to generalize biochemical composition from multiple studies. In the revised manuscript we replaced the single-study values with a generalization based on several recent reports. The revised sentence reads:“Among these, Chlorella vulgaris is recognized as one of the most promising species due to its rapid growth, resilience to diverse environments, and high-value biochemical composition. Several studies report that biomass composition typically contains proteins in the range of ~19–55% DW [17-19], lipids ~5–58% DW [20-21], carbohydrates ~10–50% DW [22-23], and pigments (chlorophylls and carotenoids) around ~1–2% DW [24-25].” This revision has been incorporated into the Introduction section and is highlighted in grey in the revised manuscript.

Reviewer’s Comment 7:

Use the correct name of the medium (Bold’s basal medium).

Response:
We thank the reviewer for pointing this out. The name of the medium has been corrected to Bold’s basal medium throughout the manuscript. This correction has been highlighted in grey in the Introduction section of the revised manuscript.

Reviewer’s Comment 8:

It is necessary to expand the list of works in the direction of modifying the composition of nutrient media with the aim of increasing the productivity of biomass and target products: Effect of nutrient supply status on biomass composition of eukaryotic green microalgae; Influence of Nutrient Medium Composition on the Redistribution of Valuable Metabolites in the Freshwater Green Alga Tetradesmus obliquus (Chlorophyta) Under Photoautotrophic Growth Conditions, and others.

Response:
We thank the reviewer for the suggestion. The manuscript has been updated to include key studies focusing on nutrient medium modification to enhance biomass and metabolite production. In particular, the studies by Procházková et al. (2014) and Ziganshina & Ziganshin (2025) were added. These additions have been highlighted in grey in the Introduction section of the revised manuscript.

Reviewer’s Comment 9:

Clarification needed on products: «The PFPP facility produces ready-to-eat meals, frozen foods, and chilled foods».

Response:
We appreciate the reviewer’s suggestion. The manuscript has been revised to provide a clearer description of the products produced at the PFPP facility in Section 2.1 (highlighted in grey). The updated sentence now reads: “The PFPP facility produces a variety of ready-to-eat meals, frozen foods, and chilled foods, including products such as steamed chicken breast, fried chicken, sausages, and other processed meat items.”

Reviewer’s Comment 10:

Sections 2.1 and 2.2 contain repetitions.

Response:

We thank the reviewer for highlighting this issue. To reduce repetition and improve clarity, Sections 2.1 and 2.2 have been revised so that Section 2.1 focuses on the collection and preparation of wastewater samples, while Section 2.2 describes the analytical procedures and preparation of wastewater for cultivation. Overlapping content was removed from Section 2.1 and incorporated appropriately into Section 2.2. These revisions have been highlighted in grey in the revised manuscript.

Reviewer’s Comment 11:

The use of autoclaving will increase the cost of the proposed biotechnology.

Response:

We thank the reviewer for this important observation. In the present study, autoclaving was applied to wastewater samples to ensure sterility under laboratory conditions. We acknowledge that autoclaving would increase costs at an industrial scale. To address this, we have added a statement in Section 2.2 (highlighted in grey).

Reviewer’s Comment 12:

The use of distilled water is not recommended; it is better to replace it with phosphate buffer: «Cells were harvested by centrifugation at 7,000 × g, 20 °C for 10 minutes, washed twice with sterile distilled water, and resuspended in wastewater».

Response:
We thank the reviewer for this helpful suggestion. The manuscript has been revised so that cells are now washed and resuspended using a phosphate buffer instead of distilled water, as recommended. This change has been incorporated in Section 2.3 and is highlighted in grey in the revised manuscript.

Reviewer’s Comment 13:

Section 2.3 should be carefully edited to avoid confusion. What was the volume of inoculum (100 mL)?

Response:
We thank the reviewer for pointing this out. To avoid confusion, Section 2.3 has been carefully edited, and the sentence “A 100 mL of this culture was transferred to a 250 mL Erlenmeyer flask” has been removed. This revision clarifies the description of inoculum preparation and avoids potential misunderstandings.

Reviewer’s Comment 14:

Plus, it is necessary to provide a rationale for choosing such a low PPFD, and provide a description of aeration, inorganic carbon supply, pH changing... the use of the applied format, 3.5-L culture bottles (shading of cells) and a fairly low PPFD level does not allow to obtain good results in terms of culture productivity (which is also evident from the level of OD and chlorophyll in the cells).

Response:

We thank the reviewer for this valuable comment. The manuscript has been revised to clarify the laboratory-scale cultivation conditions of C. vulgaris var. vulgaris TISTR 8261. The revised description in Section 2.3 (highlighted in grey) now reads: "For laboratory-scale cultivation, C. vulgaris var. vulgaris TISTR 8261 was inoculated into transparent 3.5-L glass culture bottles at a working volume of 3,000 mL, with an initial OD₇₅₀ of ~0.1. The cell cultures were cultivated at 25 ± 2 °C under continuous magnetic stirring to provide aeration and facilitate gas exchange, with white fluorescent light at an intensity of 100 μmol photons m⁻² s⁻¹ for 15 days. The pH of the cultures was measured every 2 days using a pH meter (Starter 2100, Ohaus, Parsippany, New Jersey, USA)." In addition, the corresponding results have been added in Section 3.5 (highlighted in grey): "The pH of the cultures was monitored and remained relatively stable (~7.0–7.5) throughout the cultivation period (Data not shown)."

Reviewer’s Comment 15:

Clarify: «Under cultivation in photobioreactor, C. vulgaris var. vulgaris TISTR 8261 was cultivated in 3.5-L culture bottles under controlled laboratory conditions».

Response:

Response to Reviewer Comment:
We thank the reviewer for the suggestion. The manuscript has been revised to clarify the laboratory-scale cultivation conditions of C. vulgaris var. vulgaris TISTR 8261 in 3.5-L culture bottles. The revised description in Section 2.3 (highlighted in grey) now reads:
"The cell cultures were cultivated at 25 ± 2 °C under continuous magnetic stirring to provide aeration and facilitate gas exchange, with white fluorescent light at an intensity of 100 μmol photons m⁻² s⁻¹ for 15 days."

Reviewer’s Comment 16:

Section 2.5: total chlorophyll?

Response:

We thank the reviewer for pointing this out. In this study, chlorophyll measurement was conducted according to Lee and Shen [45], where absorbance values at 664 nm and 647 nm were used to calculate total chlorophyll concentration. We have revised the section to explicitly state that the reported values represent “total chlorophyll concentration” (Section 2.5, highlighted in grey).

Reviewer’s Comment 17:

The authors should explicitly state that they verified ANOVA assumptions (Shapiro–Wilk for normality and Levene’s for homogeneity, p > 0.05) before analysis, or else indicate the use of a nonparametric alternative (e.g., Kruskal–Wallis).

Response:
We appreciate the reviewer’s valuable suggestion. In the revised manuscript, we have clarified that ANOVA assumptions were verified prior to statistical analysis. Specifically, the Shapiro–Wilk test was applied to assess normality, and Levene’s test was used to examine the homogeneity of variances. Since all results showed p > 0.05, the assumptions were satisfied, and one-way ANOVA followed by Duncan’s multiple range test was conducted. This revision has been incorporated into the Statistical Analysis section (Section 2.8, highlighted in grey).

Reviewer’s Comment 18:

What were the levels of ammonium nitrogen in the wastewater initially, before modification?

Response

We thank the reviewer for this important comment. In this study, nitrogen levels in the PFPP wastewater were analyzed in terms of total Kjeldahl nitrogen (TKN), which represents the sum of organic nitrogen and ammonium nitrogen. The initial TKN concentration in PFPP wastewater was 25.01 ± 0.15 mg L⁻¹ (Results, Section 3.1). However, ammonium nitrogen was not measured separately. Since TKN includes both organic and ammonium forms, it was used as the representative nitrogen parameter in our wastewater characterization. The supplementation with ammonium chloride in the growth optimization experiments was intended to ensure the immediate availability of inorganic nitrogen, as organic nitrogen present in the wastewater may not be readily accessible to algal cells.

Reviewer’s Comment 19:

«2.7. Nutritional Analysis of C. vulgaris var. vulgaris TISTR 8261» change to «2.7. Biomass characteristics of C. vulgaris var. vulgaris TISTR 8261»

Response:

We thank the reviewer for the helpful suggestion. We agree with the recommendation and have revised the section title accordingly “2.7. Biomass Characteristics of C. vulgaris var. vulgaris TISTR 8261” (Section 2.7, highlighted in grey).

Reviewer’s Comment 20:

Include in Table 1 the TAP composition (mg L^-1) to compare key components with their levels in alternative media.

Response:

We thank the reviewer for this constructive suggestion. We agree that including the TAP composition will provide a more meaningful comparison of nutrient levels between synthetic medium and wastewater. Accordingly, we have added TAP composition values (mg L⁻¹) to Table 1 in Section 3.1 (highlighted in grey). This allows readers to directly compare the key macro- and micronutrient concentrations between wastewater samples and the standard TAP medium.

Reviewer’s Comment 21:

Figure 4 should be replaced or supplemented to provide a complete picture of the cultivation so that readers can see how the culture was cultivated (provision of stirring, lighting, aeration...).

Response
We appreciate the reviewer’s valuable suggestion. In response, Figure 4 has been revised to include an additional panel illustrating the model of laboratory-scale cultivation in 3.5-L transparent glass culture bottles operated under controlled conditions of light and mixing (panel A). This modification provides readers with a more complete understanding of the cultivation setup, including stirring and lighting conditions (Figure 4, highlighted in grey).

Reviewer’s Comment 22:

Use “unmodified PFPP” instead of “normal PFPP” and “modified PFPP” instead of “optimal PFPP”.

Response:
We thank the reviewer for the valuable suggestion. As recommended, we have revised the terminology throughout the manuscript by replacing “normal PFPP” with “unmodified PFPP” and “optimal PFPP” with “modified PFPP”. These corrections have been consistently applied in the main text, all figures, and tables to ensure clarity and uniformity in the presentation (highlighted in grey).

Reviewer’s Comment 23:

Section 2.4 Provide the equation for biomass productivity calculation and explain which timepoints were used for SGR and BP calculation (during the exponential phase of growth?).

Response:
We appreciate the reviewer’s insightful comment. In response, we have revised Section 2.4 to include the equation for biomass productivity (BP) calculation and clarified the timepoints used for both specific growth rate (SGR) and biomass productivity determination. Specifically, SGR and BP were calculated using data points obtained during the exponential growth phase to ensure accuracy and consistency (Section 2.4, highlighted in grey).

Reviewer’s comment 24:

Based on the growth indicators, I strongly ask the authors to review the data on dry weight; it is clear that Maximum Biomass yield (g L⁻¹) and Maximum Biomass production (g L⁻¹ day⁻¹) are calculated incorrectly and raise serious doubts (even based on the intensity of the culture medium in Figure 4).

Response to Reviewer Comment:

We thank the reviewer for this careful and important observation. Following your comment, we thoroughly reviewed the raw dry-weight data, unit conversions, and calculations used to derive biomass-related indicators. As a result, the biomass productivity values (g L⁻¹ day⁻¹) have been corrected in Section 3.5 and Table 5. The revised values are highlighted in grey in the manuscript.

Reviewer’s comment 25:

Mixotrophic cultivation (with organic carbon sources) supply is creating a very high risk of contamination of the microalgae culture and obtaining inaccurate results. Please provide data about bacterial loading at the end of the cultivation period.

Response:
We appreciate the reviewer’s valuable comment. During the experiment, we carefully monitored bacterial contamination by microscopic observation and plate count at the end of the cultivation period. The results confirmed that no bacterial contamination occurred in all cultures. We have now added this information in Results section (3.2) to clarify that the mixotrophic cultivation was free from bacterial contamination (highlighted in grey).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am happy to accept the publication of this paper in its present form

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed revised manuscript and am pleased to confirm that all of my previous comments and suggestions have been thoroughly and effectively addressed.

Reviewer 4 Report

Comments and Suggestions for Authors

phycology-3854937 (Report 2)

The authors answered many questions and made the necessary corrections. However, I have a major question regarding the methodological aspects of the study, specifically the cultivation mode. Judging by the introduction and description in the study, the authors conclude that they are working with a mixotrophic cultivation mode (light as the energy source, CO₂ and organic carbon as the carbon sources), but according to 2.3 Green Algal Strain and Cultivation, and Figure 4, the algae grew without an inorganic carbon source as well as oxygen.

The second aspect that remains unanswered concerns the biomass yield. Based on the cell count and biomass yield data, the data contradict each other (Figure 5). Cell concentration values do not exceed 10⁶ cells in ml, while the biomass weight is over 8 g/L (dry weight?). The authors should include data (or subtract) concerning the biomass of bacterial cells (for cultivation in wastewater). Regarding the cultivation in TAP medium, either there was an error in the calculations or the cultivation medium was contaminated (including during sampling). Data on the bacterial load obtained by culture analysis methods should be included.