Review Reports - Co-Cultivation of <i>Chlorella vulgaris</i> Enhances Growth and Bioactive Compounds in Hydroponically Grown Lettuce

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The subject is interesting to the research community. The manuscript is generally well-written although some sentences may need improvements. My major concerns are listed below:

Phrases like "lettuce vegetable growth" and "lettuce water planting process" sound unnatural. Standard terms like "lettuce growth" and "hydroponic cultivation" should be used instead.

Line 93-105 : The word "study" or "studied" appears 5 times in a single paragraph. "Hydroponic" appears 4 times. Varying your word choice (e.g., investigation, research, system, approach) will make it sound much more professional.

Lines 123–125 contain a dangling sentence structure: "Once the lettuce seedlings had four cotyledons... the lettuce seedlings with significant differences in growth conditions, and take lettuce seedlings with suitable cultivation conditions..." This needs to be rewritten to clearly state that seedlings with uniform growth were selected while outliers were discarded.

Consolidated the baffle dimensions into a standard format (120x 60 x 30mm) to save space and read more professionally.

"Expansional culture" should be scale-up culture or mass cultivation.

"Light colour was purple" is better expressed as purple light spectrum or by stating the specific light source (e.g., LED lamps with a specialized purple spectrum).

Replaced "absorbance value was made" with "standard calibration curve was constructed." Changed "maximum absorbance at 680 nm" to "maximum absorption peak λ=680 nm."

Please include Equations 2, 3, and 4, as they are currently missing from the text.

Equations 5-9 are missing from the text; please insert them.

The conclusions must include the specific numerical values of the main results obtained.

Author Response

Reviewer 1#

Comments and Suggestions for Authors

The subject is interesting to the research community. The manuscript is generally well-written although some sentences may need improvements. My major concerns are listed below:

Response: Thanks for your professional reviews. The manuscript has been revised point by point and marked in red according to your comments.

Q1: Phrases like "lettuce vegetable growth" and "lettuce water planting process" sound unnatural. Standard terms like "lettuce growth" and "hydroponic cultivation" should be used instead.

Response: Done. The terms in line 13 and 21 have been revised.

Thanks for your professional suggestions!

Q2: Line 93-105: The word "study" or "studied" appears 5 times in a single paragraph. "Hydroponic" appears 4 times. Varying your word choice (e.g., investigation, research, system, approach) will make it sound much more professional.

Response: Done. The words have been revised as can be seen in the line 94-95, 104, 546. Thanks for your diligent review works.

Q3: Lines 123–125 contain a dangling sentence structure: "Once the lettuce seedlings had four cotyledons... the lettuce seedlings with significant differences in growth conditions, and take lettuce seedlings with suitable cultivation conditions..." This needs to be rewritten to clearly state that seedlings with uniform growth were selected while outliers were discarded.

Response: Done.

The contents were revised as,

“Lines 123–127:

In our laboratory, the species of microalgae utilized was Chlorella vulgaris, which was generously donated by the Microalgae Culture Collection Laboratory at Hainan University, China. Seeds of Italian lettuce (Lactuca sativa L., obtained from a local supplier in China) were sterilized with 70% ethanol for 45 s, then treated with a 5% bleach and sodium dodecyl sulfate (SDS) aqueous solution for 15 min, and washed 5 times with double‑deionized water. After sterilization, seeds were sown in culture trays (500⨯150⨯20 mm, custom‑made, China) containing deionized water. The lettuce seeds used in the experiment were sown in seedling trays, and the seedlings were nursed in a 26‒30 ℃ environment of about 5-days. Abnormal individuals are removed, and lettuce seedlings with four cotyledons, uniform plant height (5 ± 0.25 cm), and uniform root length (2 ± 0.1 cm) are selected and used for subsequent experiments. Green leaf growth and root vigour were adopted in the following microalgae-aided hydroponic cultivation experiments.”

Q4: Consolidated the baffle dimensions into a standard format (120x 60 x 30mm) to save space and read more professionally.

Response: Done. The dimensions have been revised. Thanks for your comments!

Q5: "Expansional culture" should be scale-up culture or mass cultivation.

Response: Done. The words have been revised as scale-up culture, as can be seen line 113. Thanks for your comments!

Q6: "Light colour was purple" is better expressed as purple light spectrum or by stating the specific light source (e.g., LED lamps with a specialized purple spectrum).

Response: Done. The light spectrum of the violet light was supplied in line 114. Thanks for your suggestions.

Q7: Replaced "absorbance value was made" with "standard calibration curve was constructed." Changed "maximum absorbance at 680 nm" to "maximum absorption peak λ=680 nm."

Response: Done. The changes were conducted as can be seen in line 186. Thanks for your comments!

Q8: Please include Equations 2, 3, and 4, as they are currently missing from the text.

Response: I am very sorry for the omitted equations. The equations 2, 3, and 4 were supplied in the revised manuscript. Thanks for your professional comments!

Q9: Equations 5-9 are missing from the text; please insert them.

Response: I am sorry for the missing of the equations. The equations 5-9 were supplied. Thanks for your comments!

Q10: The conclusions must include the specific numerical values of the main results obtained.

Response: The numerical values of the main results, including “Chlorella vulgaris nutrient solution achieved at a concentration of 1.5×10⁷ cells/mL can increase 1.5 times the total chlorophyll content in microalgae-co-cultured lettuce.”, were supplied.

Great thanks for your comments and professional suggestions. We hope the above revisions could reach your scientific review standards.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the co-cultivation of Chlorella vulgaris with hydroponically grown lettuce using aerated photobioreactor-assisted systems and evaluated the effects on plant growth, chlorophyll accumulation, root morphology, and nutritional quality parameters, such as vitamin C, soluble protein, and soluble sugar content. The manuscript requires extensive restructuring, statistical clarification, and language editing before it can be considered for publication

Abstract

Lines 11–26] The abstract is excessively descriptive and contains several grammatical errors. Quantitative results are presented without statistical context (e.g., p-values, replication, and variance). The phrase “growth rates during the 30-day proliferating of microalgae cells” is unclear and scientifically imprecise.
Lines 20–24: The claim that vitamin C levels increased by 249% requires statistical validation and comparison with absolute values. However, a biological explanation for this dramatic increase is lacking.

The abstract should clearly state the following:

experimental replication,
statistical approach,
exact treatment design,
significance level,
key mechanistic insights.

Introduction

Lines 30–91: The Introduction lacks a coherent scientific narrative. The literature review appears fragmented and frequently shifts between unrelated concepts, including hydroponics, photobioreactors, cell proliferation, root anatomy, and wastewater systems, without a strong conceptual linkage.

The authors repeatedly use the phrase “cell proliferation” in the context of lettuce growth without directly measuring cellular proliferation markers, cell cycle regulators, the mitotic index, or histological quantification. Therefore, a repeated mechanistic interpretation is not justified.

Lines 78–82: The statement regarding “cell-cycle regulators dominant the interface between cell division and differentiation” is disconnected from the actual experimental design because no molecular or transcriptomic analyses were performed.

The novelty statement should be rewritten clearly. At present, it is difficult to distinguish how this study fundamentally differs from previous hydroponic–microalgae co-cultivation studies already reported in the literature.

The Introduction should include the following:

recent literature on algal biostimulants in hydroponics,
nutrient competition between algae and plants,
oxygenation-mediated root responses,
microbial interactions in co-culture systems,
limitations of previous studies.

Materials and Methods

Lines 107–137: The description of the photobioreactor design is incomplete and difficult to reproduce experimentally. Important missing information includes the following:

reactor working volume,
hydraulic retention time,
light spectrum details,
photoperiod,
dissolved oxygen concentration,
temperature regulation,
CO₂ supplementation,
sterility conditions.

Lines 122–127: Seedling selection criteria are subjective (“significant differences”, “suitable cultivation conditions”). Authors should define objective criteria, such as biomass range or uniformity thresholds.

The manuscript lacks a clear experimental design table. The essential missing information includes the following:

number of biological replicates,
number of plants per treatment,
experimental randomization,
independent experimental repeats,
Environmental chamber specifications.

Lines 144–161: The nutrient composition of the “composite beneficial algae medium” is not clearly described. The authors should provide the following:

macro- and micronutrient concentrations,
EC values,
nutrient replenishment frequency,
nutrient depletion monitoring,
dissolved oxygen measurements.

The “free concentration” (FC) treatment is poorly defined. It remains unclear whether algal density was uncontrolled naturally or whether nutrient replenishment occurred.

No statistical analysis was performed. This is a major weakness of the study. The manuscript must explicitly state the following:

statistical software,
test used (ANOVA, Tukey HSD, etc.),
normality testing,
homogeneity testing,
significance threshold,
error representation.

Lines 180–280: Several measurement methods were inadequately described. For example:

root vigor methodology lacks calibration detail,
chlorophyll extraction equations are poorly formatted,
soluble sugar assay protocol is incomplete,
HPLC validation parameters are absent.

The manuscript does not mention whether the measurements were conducted on a fresh weight or dry weight basis consistently.

The equations are improperly formatted, missing throughout the manuscript, and several symbols are undefined or inconsistently represented. This significantly affects the readability and reproducibility.

Results

Lines 283–331: The Results section often mixes interpretation with data presentation. Biological explanations should be moved to the Discussion section.

The figures lack statistical annotations. There are no significant letters, p-values, or indications of variance comparison among treatments.

The manuscript repeatedly uses terms such as “significant increase” without providing the statistical evidence.

Figure 3 and 4: The graphical quality is inadequate for a Q1 journal. Font sizes are small, the axis labels are unclear, and the figure resolution appears low. Standard deviations and error bars are inconsistently presented.

Lines 343–357: The proposed explanation that algae adsorbed onto roots and inhibited nutrient absorption is speculative because no microscopy, biofilm quantification, or nutrient uptake assays were performed.

Root anatomical analysis is largely qualitative. Quantitative histological metrics such as:

xylem diameter,
cortex thickness,
vascular bundle number,
root cross-sectional area,
should be included.

Lines 393–401: The statement that chlorophyll increased “1.5 times” requires statistical validation and should include the exact values with variance.

The QY/Fv/Fm values are close to the optimal photosynthetic efficiency (~0.8); however, the manuscript does not adequately interpret the physiological relevance or stress implications.

Lines 465–512:
The nutrient quality section is promising but insufficiently supported. Authors should provide:

calibration curves,
chromatograms,
detection limits,
extraction efficiencies,
recovery percentages.

Discussion

The Discussion section is largely repetitive and descriptive. It frequently restates the results instead of critically interpreting the mechanisms.

The authors overemphasized “cell proliferation,” despite not directly measuring cell division. This terminology should be replaced with “growth enhancement” or “improved biomass accumulation” unless cellular analyses are included.

The manuscript does not sufficiently discuss the potential negative interactions between algae and lettuce, including the following:

nutrient competition,
shading effects,
oxygen fluctuations,
microbial contamination,
allelopathic metabolite.

The Discussion lacks integration with recent hydroponic microbiome literature and algal biostimulant mechanisms.

Several mechanistic claims remain speculative because no molecular, metabolomic, or phytohormonal analyses have been conducted.

Conclusion

Lines 560–571: The Conclusions overstate the findings. The manuscript does not demonstrate the following:

actual cost-effectiveness,
industrial scalability,
renewable energy implications,
mechanistic proof of enhanced cell division.

The conclusion should be rewritten to reflect only the experimentally demonstrated findings.

Minor Comments

12–13	“limitedly appreciated” is grammatically incorrect.
16	The phrase 30-day proliferating” should be rewritten.
26	“greener biotechnology” is vague and promotional.
38–40	The sentence structure is unclear.
48–50	The discussion of light color is disconnected from the study objectives.
56–66	An excessive engineering background dilutes the biological focus.
79–81	Unsupported reference for cell cycle regulation.
113–114	The light intensity in lux is insufficient; therefore, PPFD values should be provided.
1129	“logarithmic growth telophase” is a biologically inaccurate term.
163–172	The OD-cell density calibration lacked an R² value.
180–183	Harvest timing rationale is absent.
191–194	Leaf area estimation using the ellipse assumption may underestimate the actual area.
222–224	The equation formatting is corrupted.
289	The regression equation requires goodness-of-fit statistics.
325	“apoptosis” is inappropriate for unicellular algal culture decline.
340–341	Claims regarding phytohormone release are unsupported experimentally.
367–376	The description of root anatomy lacks quantitative rigor.
418–423	Oxygen-mediated explanations are speculative without dissolved oxygen measurements.
440	Reference to “tomato root” appears erroneous since lettuce was studied.
516–517	The sentence is grammatically incomplete.
567–569	Economic claims unsupported by techno-economic analyses.

Author Response

Reviewer 2#

Responds: We thank the reviewer for professional reviews. In the revised manuscript, we have responded the review comments, while the revised points were marked with dark red color. The following sections were

Question 1: Abstract

Lines 11–26: The abstract is excessively descriptive and contains several grammatical errors. Quantitative results are presented without statistical context (e.g., p-values, replication, and variance). The phrase “growth rates during the 30-day proliferating of microalgae cells” is unclear and scientifically imprecise.
Lines 20–24: The claim that vitamin C levels increased by 249% requires statistical validation and comparison with absolute values. However, a biological explanation for this dramatic increase is lacking.

The abstract should clearly state the following:

experimental replication,
statistical approach,
exact treatment design,
significance level,
key mechanistic insights.

Responds: I am sorry for the imperfect writing of the abstract. The p-value and revised phrases and the recheck VC contents have been throughout supplied in lines 15-27. Thanks for your reviews.

Question 2: Introduction

Responds: Great thanks for your comments which promote the deep thoughts of the introduction structure and key concepts. The manuscript has revised the introduction and supplied the necessary reference paper for the highlights of cultivation technique points including the different microalgae liquid concentration as bio-stimulant for lettuce soilless culture and the pH control strategies and device settlings. We strongly appreciate for your insightful suggestions and hope to reach the well-written standard for a better introduction section.

Question 3: Lines 78–82: The statement regarding “cell-cycle regulators dominant the interface between cell division and differentiation” is disconnected from the actual experimental design because no molecular or transcriptomic analyses were performed.

The Introduction should include the following:

recent literature on algal biostimulants in hydroponics,
nutrient competition between algae and plants,
oxygenation-mediated root responses,
microbial interactions in co-culture systems,
limitations of previous studies.

Responds: I am sorry for these issues related to the lacks of the cell-cycle regulators and molecular analyses. This study presented the findings of the microalgae cultivation technique contributions on the lettuce soilless culture. Then, the research’s key points were not put on molecular or transcriptomic analyses, but the feasible practice for the hydroponic engineering. Therefore, the descriptions on the effects of pH on cell growth and plant growth in PBR were improved, as can be seen in line 84-87.

Furthermore, responding to your scientific comments, the manuscript supplied the results analysis, including nutrient competition, oxygenation‑mediated root responses and microalgae‑mediated microbial interactions for plant growth, as can be seen line 374, 526 and 555 in the revised manuscript. The microbial interactions within the co-culture system add another layer of complexity and can be enhanced through synergistic effects on plant growth and stress tolerance were presented.

Thank you for your professional comments!

Question 4: Materials and Methods

Lines 107–137: The description of the photobioreactor design is incomplete and difficult to reproduce experimentally. Important missing information includes the following:

reactor working volume,
hydraulic retention time,
light spectrum details,
photoperiod,
dissolved oxygen concentration,
temperature regulation,
CO₂ supplementation,
sterility conditions.

Responds: We thank the reviewer for pointing out the incomplete description of the photobioreactor design. In the revised manuscript, the description of the PBR was supplied in lines 115–163.

In this research, a newly designed hydroponic system for the co-cultivation of lettuce and Chlorella vulgaris in water is shown in Figure 1. The total liquid volume in the system was 3.5 L. The main microalgae culture unit was connected to the co-cultivation device by a peristaltic pump at a flow rate of 185.0 mL/min. An aeration system equipped with a controllable airflow meter was installed in the culture tubing area, and the aeration rate was maintained at 1.0 L/min. In addition, air jet nozzles were installed in the lettuce root zone at 15.5 cm spacing to ensure full contact between the air and the algal culture solution. In this system, an LED white light (peak wavelength 460 nm) was positioned 45 cm directly above the planting basket, providing sufficient light for lettuce growth at photosynthetic photon flux density (PPFD) of 250-350 μmol·m⁻²·s⁻¹. The photoperiod was set to 16 h·d⁻¹, and the temperature was maintained at 20–25 ℃ with a relative humidity of approximately 60% throughout the cultivation period. While Chlorella vulgaris is a photoautotrophic microalga capable of utilizing ambient CO₂ for photosynthesis, the aeration rate (1.0 L/min) was set in this study, which ensures sufficient gas exchange with additional CO₂ enrichment.

The sterility conditions were supplied, and the microalgal inoculum was maintained under axenic conditions prior to inoculation to ensure a pure starting culture. However, the co‑cultivation system itself was not kept strictly sterile. The primary reason is that achieving absolute sterility is technically unattainable in an open hydroponic system over a 30‑day cultivation period, as lettuce roots naturally carry surface‑associated microorganisms, and the circulating nutrient solution inevitably becomes colonized by environmental microbes. Strict sterility has not been performed for the purpose of this study because the presence of the actual background microorganisms better reflects realistic hydroponic production conditions, while the analysis of the algae-plant interactions was provided in the revised manuscript.

Question 5: Lines 122–127: Seedling selection criteria are subjective (“significant differences”, “suitable cultivation conditions”). Authors should define objective criteria, such as biomass range or uniformity thresholds.

The manuscript lacks a clear experimental design table. The essential missing information includes the following:

number of biological replicates,
number of plants per treatment,
experimental randomization,
independent experimental repeats,
Environmental chamber specifications.

Responds: We thank the reviewer for raising the important points regarding the objectivity of seedling selection and the missing experimental design information. In response, we have supplemented the manuscript accordingly:

Seedling selection section

Lines 126–137:

The species of microalgae utilized was Chlorella vulgaris, which was generously donated by the Microalgae Culture Collection Laboratory at Hainan University, China. Seeds of Italian lettuce (Lactuca sativa L., obtained from a local supplier in China) were sterilized with 70% ethanol for 45 s, then treated with a 5% bleach and SDS (Sodium Dodecyl Sulfate) aqueous solution for 15 min, and washed 5 times with double‑deionized water. After sterilization, seeds were sown in culture trays (500⨯150⨯20 mm, custom‑made, China) containing deionized water. The lettuce seeds used in the experiment were sown in seedling trays, and the seedlings were nursed in a 26‒30 ℃ environment of about 5-days. Abnormal individuals are removed, and lettuce seedlings with four cotyledons, uniform plant height (5 ± 0.25 cm), and uniform root length (2 ± 0.1 cm) are selected and used for subsequent experiments. Green leaf growth and root vigor were adopted in the following microalgae-aided hydroponic cultivation experiments.

Experimental design section

Lines 148–159:

In this research, a newly designed hydroponic system for the co-cultivation of lettuce and Chlorella vulgaris in water is shown in Figure 1. The total liquid volume in the system was 3.5 L. The main microalgae culture unit was connected to the co-cultivation device by a peristaltic pump at a flow rate of 185.0 mL/min. An aeration system equipped with a controllable airflow meter was installed in the culture tubing area, and the aeration rate was maintained at 1.0 L/min. In addition, air jet nozzles were installed in the lettuce root zone at 15.5 cm spacing to ensure full contact between the air and the algal culture solution. In this system, an LED white light was positioned 45 cm directly above the planting basket, providing sufficient light for lettuce growth at photosynthetic photon flux density (PPFD) of μmol·m⁻²·s⁻¹. The photoperiod was set to 16 h·d⁻¹, and the temperature was maintained at 20–25 ℃ with a relative humidity of approximately 60% throughout the cultivation period.

Lines 180–184:

Eight lettuce plants in each system were grown in each experiment, repeated three times. All plants were harvested 30 days after transplanting. Three lettuce plants were randomly selected from each growth system for subsequent measurements.

Question 6: Lines 144–161: The nutrient composition of the “composite beneficial algae medium” is not clearly described. The authors should provide the following:

macro- and micronutrient concentrations,
EC values,
nutrient replenishment frequency,
nutrient depletion monitoring,
dissolved oxygen measurements.

The “free concentration” (FC) treatment is poorly defined. It remains unclear whether algal density was uncontrolled naturally or whether nutrient replenishment occurred.

No statistical analysis was performed. This is a major weakness of the study. The manuscript must explicitly state the following:

statistical software,
test used (ANOVA, Tukey HSD, etc.),
normality testing,
homogeneity testing,
significance threshold,
error representation.

Responds: Thank you for your professional comments. We have provided detailed information on the nutrient composition of the "composite beneficial algae medium" and have supplied the statistical analysis in the manuscript. In the revised manuscript, we will add a complete "Statistical Analysis" subsection, which will specifically include. We thank the reviewer for pointing out the lack of clarity regarding the free concentration (FC) treatment. To address this, we have now clearly defined the FC treatment. We hope this definition resolves any ambiguity about whether algal density was uncontrolled and whether nutrients were replenished. The corresponding description in the revised manuscript is as follows, please find this section directly below:

Lines 160–162:

The hydroponic containers were prepared and filled with a culture solution using the composite beneficial algae medium as the substrate (initial pH of 7.08 ± 0.22, electrical conductivity of 0.964 ± 0.033 mS·cm⁻¹) (see Table S1 for nutrient composition in the supplementary information).

Table S1 Composition lists of medium

Lines 168–177:

One treatment group, the free concentration (FC) group, was designated as the culture group. Under this treatment condition, the FC group initially contained a low concentration of Chlorella sp. After the initial algal inoculation, no artificial density adjustment was performed, and the algae were allowed to grow naturally. Meanwhile, two pure nutrient solution groups (NS group-commercial nutrient solution cultivation and PW group-pure water cultivation) were used as control groups.

The selected lettuce seedlings had similar growth conditions in Section 2.1, were placed in hydroponic cotton and transplanted into the apparatus's planting basket for 30 days of co-cultivation. Every 7 days, they supplemented these systems with 0.25 L of culture medium.

Lines 347–352:

2.6 Statistical analysis

All experiment were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was conducted using SPSS 26.0 software, and one-way analysis of variance (ANOVA) was employed to evaluate differences between groups. A probability value of P<0.05 was considered statistically significant. Root section images were further processed using Case Viewer software, and data plotting was performed using Origin 2024 software.

Question 7: Lines 180–280: Several measurement methods were inadequately described. For example:

root vigor methodology lacks calibration detail,
chlorophyll extraction equations are poorly formatted,
soluble sugar assay protocol is incomplete,
HPLC validation parameters are absent.

The manuscript does not mention whether the measurements were conducted on a fresh weight or dry weight basis consistently.

Responds: Thanks for the reviewer's comments and we have revised the manuscript accordingly, adding details as follows:

Lines 228–243: (root vigor)

Measurement of total absorption area, active absorption area, and percentage of active absorption area of lettuce roots in different treatment groups. A methylene blue solution (0.01 mg/mL) was prepared and further diluted with deionised water to obtain a series of gradient concentration standards. The absorbance of each standard solution was measured at 660 nm using a spectrophotometer, and a standard curve was constructed by plotting the absorbance values.

Lettuce seedlings were randomly selected from each treatment group. The roots were carefully washed and blotted dry with absorbent paper to remove surface moisture. Root volume was determined using the water displacement method. Three small beakers were prepared, each containing 0.0002 mol/L methylene blue solution, equal to 10 times the measured root volume. The root system was sequentially immersed in the three beakers for 1.5 min per beaker. Then, 1 mL of the methylene blue solution from each beaker was mixed with deionised water at a 1:9 (methylene blue: water, v/v) ratio. The mixture was vortexed, and its absorbance was measured at 660 nm. The concentration of methylene blue after root immersion was calculated using the standard regression equation obtained from the experiment. The following formulas were used for calculation:

Lines 286–297: (HPLC validation parameters)

VC standard solutions at gradient concentrations were prepared using 0.1% oxalic acid solution. Each standard solution was filtered through a 0.22 μm membrane filter to obtain the standards, which were then sequentially analysed by high-performance liquid chromatography (HPLC). A standard curve was constructed by plotting the mass concentration of the VC standard solutions against the corresponding peak areas.

From each treatment group, lettuce plants were randomly selected, washed, drained, and cut into small pieces. The pieces were placed in a homogeniser, and 5 mL of 0.1% oxalic acid was added as the extraction solvent. The mixture was homogenised and filtered under vacuum. The resulting filtrate was the sample VC solution. A 5 g aliquot of the above filtrate was taken and diluted tenfold with 0.1% oxalic acid. Prior to analysis, the sample solution was also filtered through a 0.22 μm membrane filter and then analysed by HPLC under the same chromatographic conditions. The VC content was calculated using the standard regression equation obtained from the experiment.

Lines 323–339: (soluble sugar)

A series of glucose standard solutions was prepared at gradient concentrations. Phenol solution was added to each, then concentrated sulfuric acid was added immediately. The mixture was shaken to ensure uniform mixing. The final solution volume was 4 mL, and the ratio of standard solution: phenol solution: concentrated sulfuric acid was 2:1:5 (v/v/v). After standing at room temperature for 20 min, the absorbance was measured at 470 nm, and a standard curve for glucose was constructed.

From each treatment group, a random sample of lettuce leaves was selected. After washing, the leaves were blotted dry to remove surface moisture, and the veins were removed. A 0.5 g sample of the treated leaf tissue was weighed, cut into small pieces, and homogenised. The resulting solution was transferred to a test tube. The remaining residue was rinsed with deionised water, and the rinsate was combined with the initial solution to obtain the sample's soluble sugar extract. Then, 1 mL of the extract was transferred to a test tube and subjected to the same treatment: phenolThen, 1 mL of the extract was transferred to a test tube and subjected to the same treatment: phenol solution was added to the extract first, followed by concentrated sulfuric acid; the mixture was shaken and allowed to stand before absorbance was measured at 470 nm.s determined from the standard curve. The following formula was used for the calculation:

We thank the reviewer for pointing out these issues. All measurements were consistently conducted on a fresh weight basis, and this has now been clearly stated in the revised manuscript. We sincerely apologize for the improperly formatted equations, which significantly affected readability. In the revised manuscript, all equations have been corrected and inserted in the appropriate sections. The revised contents are as follows:

Question 8: Results

Lines 283–331: The Results section often mixes interpretation with data presentation. Biological explanations should be moved to the Discussion section.

The figures lack statistical annotations. There are no significant letters, p-values, or indications of variance comparison among treatments.

The manuscript repeatedly uses terms such as “significant increase” without providing the statistical evidence.

Response: We thank the reviewer for the comments. Statistical analyses were originally performed but not clearly presented in the manuscript. In the revised version, all figures have been regenerated with statistical annotations (p < 0.05) added, and only data reporting is kept in the Results section. All claims of significance are now supported by statistical evidence.

Question 9: Figure 3 and 4: The graphical quality is inadequate for a Q1 journal. Font sizes are small, the axis labels are unclear, and the figure resolution appears low. Standard deviations and error bars are inconsistently presented.

Response: The figures have been updated. Thanks for your comments.

Lines 449:

Lines 505:

Question 10: Lines 343–357: The proposed explanation that algae adsorbed onto roots and inhibited nutrient absorption is speculative because no microscopy, biofilm quantification, or nutrient uptake assays were performed.

Root anatomical analysis is largely qualitative. Quantitative histological metrics such as:

xylem diameter,
cortex thickness,
vascular bundle number,
root cross-sectional area,
should be included.

Response: We thank the reviewer for these critical and constructive comments. Regarding the speculative explanation of algal adsorption inhibiting nutrient absorption, we fully agree with the reviewer that the proposed explanation was speculative. Instead of overinterpreting the mechanism, we now simply report the observed differences in root morphology and growth, and have rephrased the explanation to avoid unsupported claims. We have also added a sentence acknowledging this limitation and suggesting that future studies address these questions using appropriate imaging and nutrient flux measurements. We agree that quantitative histological metrics are necessary for rigorous root anatomy assessment. In the revised manuscript, we have now measured and included the following quantitative parameters: epidermis thickness, cortex thickness, xylem diameter, root cross-sectional area, and vascular bundle number. The data are presented in a new table (shown below) .

Lines 549: Table 1 Root and stem anatomical characteristics of cultivation groups

Root anatomy	HC	LC	FC	NS
Epidermis thickness (μm)	10.9±1.3ab	10.0±0.7b	11.7±0.5a	10.4±0.5ab
cortex thickness (μm)	165.0±23.2a	149.1±19.9b	175.7±2.8a	144.7±0.8b
xylem diameter (μm)	142.0±10.0a	159.7±31.9a	177.9±1.7a	148.8±2.0a
root cross-sectional area (μm²)	231203.4 ±31151.3b	237832.9 ±25775.6b	309896.6 ±7255.4a	228098.5 ±12641.5b
vascular bundle number	3-4	3-4	3-5	3-4

Lines 538–552:

As shown in Table 1, the root cross-sectional area (309,896.6 ± 7,255.4 μm²) and cortex thickness (175.7 ± 2.8 μm) in the FC group (high‑concentration Chlorella vulgaris) were significantly greater than those in the LC, HC, and NS groups (p < 0.05). No significant differences in xylem diameter were observed among treatments; however, the FC group exhibited the highest value. The number of vascular bundles ranged from 3 to 5 in the FC group and from 3 to 4 in the other groups. Collectively, the high‑concentration Chlorella vulgaris treatment promoted lateral root expansion and cortical thickening and tended to increase vascular bundle number, whereas the FC (free concentration) and NS groups showed lower values across several parameters. These quantitative data provide an objective foundation for understanding the effects of different co‑cultivation strategies on the root structure of hydroponic lettuce.

Question 11: Lines 393–401: The statement that chlorophyll increased “1.5 times” requires statistical validation and should include the exact values with variance.

The QY/Fv/Fm values are close to the optimal photosynthetic efficiency (~0.8); however, the manuscript does not adequately interpret the physiological relevance or stress implications.

Response: We thank the reviewer for this important comment. We agree the statement must be supported by statistical validation and exact values with variance. In the revised manuscript, we have now provided the complete statistical information as follows:

Lines 461–471:

Meanwhile, the total chlorophyll content of hydroponically grown lettuce varied significantly among treatments (p < 0.05). The highest total chlorophyll content was observed in the HC group (0.9402 ± 0.068 mg/g), which was significantly higher than all other groups. The LC group (0.7938 ± 0.044 mg/g) and FC group (0.7711 ± 0.026 mg/g) showed intermediate values without significant difference from each other, while the NS group (0.6274 ± 0.036 mg/g) had the lowest total chlorophyll content. Compared with the NS control, the HC treatment increased total chlorophyll content by approximately 1.5‑fold. Among the treatments, the optimal effect was achieved at a Chlorella vulgaris concentration of 1.5 × 10⁷ cells/mL (HC). These results indicate that a suitable concentration of Chlorella vulgaris can significantly enhance chlorophyll accumulation in hydroponic lettuce.

Question 12: Lines 465–512:
The nutrient quality section is promising but insufficiently supported. Authors should provide:

calibration curves,
chromatograms,
detection limits,
extraction efficiencies,
recovery percentages.

Responses: Thank you for your valuable comments. We fully recognize that the supporting data in the nutrient quality section were indeed insufficient. In the revised manuscript, we have supplemented the calibration curve and a representative chromatogram for vitamin C determination. During our study, we performed blank solvent injections and fitted the peak area at approximately 4.9 min, confirming that this peak corresponds to vitamin C in the liquid chromatogram. The method used in this study mainly followed that of Wang et al.^[1], which provides complete chromatographic conditions and validation parameters. Since this method has been published as a well-established approach, and the sample matrix in our study shares certain similarities with that in the reference, we directly adopted the chromatographic conditions without independently determining the detection limits, extraction efficiencies, or recovery percentages. We fully understand the importance of these parameters for method validation, as pointed out by the reviewer, and in future studies we will conduct comprehensive method validation specifically for our sample matrix to further improve the rigor of our data. Once again, we thank the reviewer for the thorough and constructive guidance.

calibration curves,

chromatograms,

Reference

[1] Wang Hui，Liu Hui，Liu Jia，Lv Du，Li Jun，Chen Chao-jun，Dong Nan， Tang Jian-bo，Chen Zhong-ai，Liu Yong-xiang. Determination of Vitamin C in Colorful Potatoes by High Performance Liquid Chromatography. Food Research and Development 2017,38(12): 130-134.

Question 13: Discussion

The Discussion section is largely repetitive and descriptive. It frequently restates the results instead of critically interpreting the mechanisms.

The manuscript does not sufficiently discuss the potential negative interactions between algae and lettuce, including the following:

nutrient competition,
shading effects,
oxygen fluctuations,
microbial contamination,
allelopathic metabolite.

The Discussion lacks integration with recent hydroponic microbiome literature and algal biostimulant mechanisms.

Several mechanistic claims remain speculative because no molecular, metabolomic, or phytohormonal analyses have been conducted.

Responses: The over discussed words of cell proliferation have been revised. The microalgae bio-stimulant mechanisms have been supplied in the descriptions on the growth enhancement facts.

Great thanks for your professional comments.

Question 14: Conclusion

Lines 560–571: The Conclusions overstate the findings. The manuscript does not demonstrate the following:

actual cost-effectiveness,
industrial scalability,
renewable energy implications,
mechanistic proof of enhanced cell division.

The conclusion should be rewritten to reflect only the experimentally demonstrated findings.

Responses: Thanks for your comments. The descriptions of the cost, industrial scalability, energy and cell division have been removed from the revised manuscript. The actual numerical data of the growth enhancement by the microalgae cultivation technique on the lettuce hydroponic systems have been concluded.

Your scientific comments help the revisions of the manuscript, and the reached to an improvement of the scientific level. We hope the revisions could reach your professional standards.

Minor Comments

12–13	“limitedly appreciated” is grammatically incorrect.
16	The phrase 30-day proliferating” should be rewritten.
26	“greener biotechnology” is vague and promotional.
38–40	The sentence structure is unclear.
48–50	The discussion of light color is disconnected from the study objectives.
56–66	An excessive engineering background dilutes the biological focus.
79–81	Unsupported reference for cell cycle regulation.
113–114	The light intensity in lux is insufficient; therefore, PPFD values should be provided.
1129	“logarithmic growth telophase” is a biologically inaccurate term.
163–172	The OD-cell density calibration lacked an R² value.
180–183	Harvest timing rationale is absent.
191–194	Leaf area estimation using the ellipse assumption may underestimate the actual area.
222–224	The equation formatting is corrupted.
289	The regression equation requires goodness-of-fit statistics.
325	“apoptosis” is inappropriate for unicellular algal culture decline.
340–341	Claims regarding phytohormone release are unsupported experimentally.
367–376	The description of root anatomy lacks quantitative rigor.
418–423	Oxygen-mediated explanations are speculative without dissolved oxygen measurements.
440	Reference to “tomato root” appears erroneous since lettuce was studied.
516–517	The sentence is grammatically incomplete.
567–569	Economic claims unsupported by techno-economic analyses.

Responses: Done. These minor comments have been point-by-point checked and revised in the manuscript.

Great thanks for your diligent review work, which benefits the improvement of the scientific manuscript!

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has improved substantially compared with the previous version. Several important methodological details have been added. The revised figures are clearer and the addition of quantitative root anatomy data strengthens the study. However, several issues still require attention before the manuscript can be considered for publication.

Major comments

The manuscript states that one-way ANOVA was performed. However, the post hoc multiple comparison procedure used to generate the significance letters shown in the figures and tables is not specified. Please indicate whether Tukey's HSD, Duncan's multiple range test, LSD, or another post hoc method was used. In addition, the manuscript does not report whether assumptions of ANOVA were evaluated (normality and homogeneity of variance tests). These details should be included in the Statistical Analysis section.

Several interpretations remain speculative and are not supported by direct measurements, including oxygenation-mediated root responses, microbial interactions within the co-culture system, hormone-mediated regulation, and enhanced nutrient transport mechanisms.

Several paragraphs in the Results section discuss potential mechanisms, nutrient competition, microbial interactions, and oxygen dynamics. These interpretations should be moved to the Discussion section, while the Results section should focus on objective presentation of experimental observations.

The HPLC methodology remains limited, the authors should provide calibration curve statistics (R²), linearity range, limit of detection (LOD), limit of quantification (LOQ), and recovery or repeatability data.

The term "cell proliferation" should be removed throughout the manuscript. The study does not directly assess cell division, mitotic activity, cell-cycle regulation, or molecular markers of proliferation. Therefore, terms such as "growth enhancement," "biomass accumulation," or "growth promotion" are more appropriate.

Minor comments

Some grammatical and stylistic issues remain throughout the manuscript. Careful professional English editing is recommended before publication.

Please ensure consistency in terminology, particularly for treatment abbreviations (HC, LC, FC, NS, PW) and their descriptions throughout the manuscript.

Author Response

Dear reviewer 2,

Thank you for the positive assessment of the improvements made in the revised manuscript and for the constructive comments that further help us improve the quality of the paper. We have addressed the issues point by point as the attachement PDF.

Author Response File: Author Response.pdf