Effect of Light Intensity on the Growth and Nutrient Uptake of the Microalga Chlorella sorokiniana Cultivated in Biogas Plant Digestate
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript examines the effect of light intensities on the growth of Chlorella sorokiniana and its role in nutrient reduction during microalgae growth. It also explores the relationship between light intensities and the lipid, protein, and carbohydrate content in microalgae biomass. However, I believe the area of study is already well-explored, and the manuscript does not present any significant new findings. Numerous published articles already cover the impact of light intensities on microalgae growth and phycoremediation. Additionally, the use of Chlorella sorokiniana, a commercial species extensively studied (e.g., Psachoulia et al., 2021, Water; Montoya-Vallejo et al., 2023, Front Bioeng Biotechnol; Usman et al., 2024, Bioresource Technology; Papapanagiotou et al., 2024, Algal Research), limits the novelty of this work.
Some of the results and discussion presented in the manuscript may be misleading or require clarification. All figures presented without any error bars, which is not meet to the journal standard. However, the manuscript could be considered if the authors can clearly demonstrate significant results that are unique to this study and make the necessary corrections.
This is my specific comments:
1. Abstract: Many other wastewater sources have a higher nitrogen content than 5 g/L. I suggest rephrasing the phrase "up to 5 g/L," as this value is relatively low. Additionally, please clarify whether this refers to total nitrogen or ammonia.
2. Introduction: It is important to explain the justification for the parameters relevant to this study, rather than providing general information. I suggest that the authors specify the rationale for using anaerobic effluent from an anaerobic digestion plant. What challenges are associated with this source, and what are the bottlenecks in the industry that make microalgae a suitable candidate for treatment? Why did the authors choose light intensities of 1500, 5000, and 12,000 lux? Why was Chlorella sorokiniana selected?
3. Line 87: The statement "However, there is no clear consensus on lipid and light intensities" is misleading, as many published articles already address this topic.
4. Materials and Methods: If the authors used a light-dark cycle of 16:8 hours, it should be mentioned in the abstract, as light intensity and duration are important factors. Please clarify why this cycle was chosen.
5. Effluent Characterization: The characterization of the effluent sample (raw and after pretreatment), including COD, BOD, total nitrogen, total phosphorus, pH, suspended solids, and turbidity, is necessary, as it will affect the reproducibility of the data in the future.
6. Section 2.3: The optical density (OD) for algae is typically measured at a wavelength higher than 600 nm, usually around 700-750 nm, to align with the absorption peak of chlorophyll. The authors should either use biomass measurements or provide a justification for using 600 nm.
7. Section 2.5.3: The intensity used for sonication method should be specified.
8. Figure 2: The macromolecule results for lipids, carbohydrates, and proteins in figures a, b, and c appear similar. The authors mention an increase in lipids with light intensity, but the graphs presented may not reflect the correct results. Please verify the data.
9. Figure 4: It is unclear why macronutrients were investigated on day 15, as this is when the nutrient content was adjusted. Please provide a rationale for this timing.
10. Statistical Analysis: I suggest adding statistical analysis for the presented data to ensure the reliability of the results.
Comments on the Quality of English Language
The manuscript requires English editing and proofreading to meet the journal's standards.
There is a spelling mistake as well.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper by Palikrousis et al. is dedicated to the investigation of the light intensity efffect on the growth of and nitrogen sequestration by the culture of Chlorella sorokiniana. Although the text is generally well-written, it is not possible to consider this manuscript for publication since it lacks the inducation of the number of biological replicas as well as statististical treatment of the data. In addition to this, the results and conclusions are trivial: the facts that culture growth depends on incident light intensity, and the nitrogen uptake depends on cell density and light intensity is well known. Furthermore, the selection of light intensities is not substantiated e.g. by light curve analysis. Luxes are deprecated units, moles of PAR quanta per unit area and time should be used instead.
Comments on the Quality of English LanguageA moderate correction of the English style should be carried out.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
your manuscript is interesting, and generally written correctly. However, to be of full value it is necessary to supplement Chapter 2 with detailed information on the fermentation effluents used.
Undoubtedly, light is a very important factor in the growth of Clorella sorokiniana, but the other important component is the nutrient content of the water. Owning this element is missing.
Supplement the chemical composition of the fermentation wastewater and necessarily improve the results and summary.
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThis study was to examine the effect of light intensity on C. sorokinianana microalgae species, cultivated in anaerobic digestate effluent. The manuscript needs to be revised before publication.
1.The abstract should include key data from the research results.
2.The significance of the study should be stated at the end of the abstract.
3.L124: Was the algae isolated from wastewater?
4.L144: Please include measurements of various water quality parameters of ADE such as pH, COD, TN, TP, etc.
5.L183: Add the formula used to calculate biomass.
6.The method for determining nutrient content is missing.
7.The section on experimental methods should include the data analysis method under "Statistical analysis."
8.In the Results and Discussion section, please describe the experimental results first, followed by the discussion.
9.The results in Figure 2 do not show any significant differences. A separate comparison of each parameter should be conducted to determine if there is a significant difference.
10.Why was the COD concentration not measured? Is the algae purely autotrophic?
11.Why do none of the figures have error bars? Was the experiment conducted in triplicate?
12.L439：”Figure 5.“
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have answered all my comments accordingly.
Author Response
Thank you very much for your valuable contribution to the revision of our manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors had dealt with the most serious issues raised by the reviewer. Still. the results are of limited novelty, but I leave the final assessment of this at the discretion of the academic editor.
Comments on the Quality of English LanguageThe English quality is satisfactory.
Author Response
We have made appropriate changes to the manuscript to highlight the novelty and importance of our research:
L(78-81)“These varying results suggest that light intensity has different effects on microalgal species, making it essential to determine optimal light parameters under well-defined conditions for each species and nutrient type [10].”
L(94-96): “Therefore, this study aimed to investigate the effect of light intensity on the production of all three macronutrients from C. sorokiniana biomass, a topic that has been underexplored in the literature.”
L(113-115): “However, effluents may contain various compounds that could affect microalgal growth patterns and composition, making it challenging to predict the effect of light intensity on the species.”
L(121-125): “This study sought to determine C. sorokiniana production rates under different light intensities, investigate nutrient assimilation rates, and assess how light intensity affects protein, lipid, and carbohydrate concentrations in the biomass. This comprehensive approach is necessary, as the simultaneous study of all macronutrients has been underexplored in the literature.”
Reviewer 4 Report
Comments and Suggestions for AuthorsThe paper has seen improvement after revisions. However, the abstract still requires further modification. The abstract should succinctly summarize the main objectives, methodologies, key findings, and implications of the research. Once the abstract is revised appropriately, the paper can be accepted.
Author Response
Thank you for your comment. We have completely changed the abstract as follows: This study investigates the effect of light intensity on the growth and nutrient uptake of Chlorella sorokiniana cultivated in nitrogen-rich anaerobic digestion wastewater. Three light intensities (20, 68, and 162 µmol m⁻² s⁻¹) were applied over a 30-day period with a 16:8-hour light-dark photoperiod. The goal was to understand how light affects biomass productivity, nutrient assimilation, and biochemical composition under varying nitrogen concentrations originating from biogas plant digestate, up to 5 g L⁻¹. Results showed that higher light intensities significantly boosted biomass production, achieving a fivefold increase at 162 µmol m⁻² s⁻¹ compared to 20 µmol m⁻² s⁻¹. Nutrient uptake followed a similar pattern, with 94% of ammonium nitrogen removed in 7 days under high light, compared to 55% after 30 days under low light. Phosphorus content was also completely removed after 7 days under light intensities of 68 and 162 µmol m⁻² s⁻¹. Additionally, elevated light intensity led to increased lipid accumulation (from 29.7% to 34%) and reduced protein content (from 30.9% to 26.1%), with carbohydrate content not being affected by light intensity. These findings highlight light intensity as a critical factor for optimizing microalgae cultivation nitrogen-rich biogas digestate, promoting both effective nutrient removal and biomass production for potential bioenergy applications.
We hope that this change fulfills your expectations. Thank you again also for your previous comments that helped us to improve our manuscript.
