Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article summarizes enhancing lipid content of Chlorella sp. by the limitation of phosphorus (P) and addition of various heavy metals. Because of the prominent nature of efforts to obtain biofuels with greener technologies instead of fossil-based fuels, this study will make a significant contribution to the field and will attract the attention of researchers from a wide range of fields.
However, there are several minor issues that prevent the publication of the work in its current form:
1. The English is poor, and correction of the Manuscript by a native speaker is recommended. As an example, the following sentence from Introduction (paragraph 2):

“Microalgae are considered to be more advantageous than crops, which are mainly due to higher growth rate, zero CO₂ producing and no need of arable lands for cell growth.”

must be corrected as:

“Microalgae are considered to be more advantageous than plant products because they have higher growth rates, produce zero CO₂, and do not require arable land for cell growth.”

Following sentence (paragraph 3):

“For Pb, which is absent in BG11medium, the study on its effect combined with P limitation is still unavailable.”

must be corrected as:

“There is no study yet examining the effect of Pb, which is not present in the BG11 medium, in combination with P limitation.”

2. Authors must comment on the inconsistencies between the data obtained for the control group for different data sets, which essentially correspond to the same physical conditions. For example, there is no increase in biomass in Figure 1A for 100% P for Day 3. However, biomass in Figure 2A almost doubles for 100%P within 3 days. Gradual increase of biomass is observed in Figure 3A for the same conditions, namely 100% P.
3. Legend of Table 1, “^a Phosphate concentration.” must be replaced with “^a Phosphorus concentration.”
4. Please add description of NA.

Comments on the Quality of English Language

The English is poor, and correction of the Manuscript by a native speaker is recommended. As an example, the following sentence from Introduction (paragraph 2):

“Microalgae are considered to be more advantageous than crops, which are mainly due to higher growth rate, zero CO2 producing and no need of arable lands for cell growth.”

must be corrected as:

“Microalgae are considered to be more advantageous than plant products because they have higher growth rates, produce zero CO2, and do not require arable land for cell growth.”

Following sentence (paragraph 3):

“For Pb, which is absent in BG11medium, the study on its effect combined with P limitation is still unavailable.”

must be corrected as:

“There is no study yet examining the effect of Pb, which is not present in the BG11 medium, in combination with P limitation.”

Author Response

This article summarizes enhancing lipid content of Chlorella sp. by the limitation of phosphorus (P) and addition of various heavy metals. Because of the prominent nature of efforts to obtain biofuels with greener technologies instead of fossil-based fuels, this study will make a significant contribution to the field and will attract the attention of researchers from a wide range of fields.

Response: Thanks for encouraging comments.

1. The English is poor, and correction of the Manuscript by a native speaker is recommended. As an example, the following sentence from Introduction (paragraph 2):

“Microalgae are considered to be more advantageous than crops, which are mainly due to higher growth rate, zero CO₂ producing and no need of arable lands for cell growth.”

must be corrected as:

“Microalgae are considered to be more advantageous than plant products because they have higher growth rates, produce zero CO₂, and do not require arable land for cell growth.”

Response: The correction has been made (Lines 47-48).

“For Pb, which is absent in BG11medium, the study on its effect combined with P limitation is still unavailable.”

must be corrected as:

“There is no study yet examining the effect of Pb, which is not present in the BG11 medium, in combination with P limitation.”

Response: The correction has been made (Lines 71-73).

The entire manuscript has been thoroughly checked and edited for English with the help of a native speaker.

2. Authors must comment on the inconsistencies between the data obtained for the control group for different data sets, which essentially correspond to the same physical conditions. For example, there is no increase in biomass in Figure 1A for 100% P for Day 3. However, biomass in Figure 2A almost doubles for 100%P within 3 days. Gradual increase of biomass is observed in Figure 3A for the same conditions, namely 100% P.

 

Response: Thanks for bringing this point to our attention.  The difference between Fig. 1A and Fig. 2A, despite the same condition with 100 % P, for the change in biomass is probably due the difference in biomass at the starting point at day 0. In Fig. 1A, biomass at day 0 is about 0.2 g/l which is about 2-fold higher than that in Fig. 2A (about 0.1 g/l). The high initial biomass might cause the delay in growth up to 3 days, which might cause the long period of the lag phase of cell growth. In

Fig. 3A, the low initial biomass enabled slow growth up to the first 3 days.

 

3. Legend of Table 1, “^a Phosphate concentration.” must be replaced with “^a Phosphorus concentration.”

Response: The correction has been made (Line 458).

 

4. Please add description of NA.

Response: The description of NA has been made (Line 458).

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

1. Scientific and practical strengths:

Topic selection is very relevant, involving the production of biodiesel from microalgae, specifically Chlorella  sp., and is consistent with present day trends of renewable energy production and greenhouse gas reduction. This research presents an integrated effect of phosphorus limitation and metal stress, which is less addressed in the literature, providing insights into the metabolism of lipids and tolerance of stress of microalgae.

It is deliberately set up as an experiment that integrates nutrient constraint (phosphorus) into toxic metal treatment (Fe, Co, Pb). It provides detailed physiological outcomes, supporting the validity and significance of outcomes for the research community.

An important aspect is the characterization of lipids and biodiesel on several dimensions. Whereas the paper does measure the content of the lipids, it proceeds to analyze fatty acid composition, and for those relevant biodiesel properties, makes conscientious comparison to astm and en standards. This further enhances the work's applied relevance.

Importantly, the authors also look at markers of oxidative stress (H₂O₂, MDA) as well as protective enzymatic activities (SOD, CAT), adding richness to physiological understanding and suggestive of a possible mechanism whereby Pb stress could impact lipid metabolism.

2 Key constraints to be met for Q1 preparedness:

A key limitation is the lack of molecular-level mechanistic analysis. The explanations for lipid accumulation remain observational, with no detailed exploration of specific lipid metabolic pathways or regulatory genes (e.g., DGAT, PDAT). In the context of current research, molecular insights are essential for elevating the manuscript’s scientific merit.

Novelty is also quite incremental. As the study presents heavy metal stress to the standard nutrient deprivation model, that pairing, while valid, lacks the disruptive impact typically desired for Q1 articles. What is presented is not significantly different from existing knowledge or technique.

Not enough has been discussed regarding the long-term toxicity and accumulation potential of heavy metals. Whereas short-term enhancement of lipids is illustrated, there is no adequate consideration of the possible ecological as well as commercial consequences of residual heavy metals in biomass.

It only studied one strain of Chlorella , limiting generalizability. It is unknown, without cross-strain comparison, whether reported effects are strain-specific or universally true for other species of microalgae.

 

3.. Recommendations for improvement: in particular:

In order to attain Q1 quality, authors are recommended to:

• Additional molecular characterization, such as rt-qpcr, transcriptomics, proteomics, or key enzyme assays (e.g., DGAT, PDAT, ACCASE), can be integrated to present an explanation of cellular processes of lipid accumulation.
• To quantify the heavy metal loading of algal cells and determine the toxicity and environmental effects of the post-treatment biomass, thereby delineating risks from benefits.
• Expand the study to include multiple Chlorella strains or related microalgae to improve generalizability.
• Provide long-term data to assess continued lipid productivity under longer-lasting pressure conditions, providing an indicator of scalability for pilot or commercial uses.

4. General conclusion:

The paper shows an adequate course with good experimental results and good practical significance. Nevertheless, as of now, it does not exhibit mechanistic profundity or unique innovation typical of leading-edge journals. It is adequate for mid-tier journals Q2–Q3, but for consideration for high-impact Q1 journals, there is a need for the authors to include extensive molecular and mechanistic investigations, expand comparative datasets, and cover heavy metal accumulation hazard thoroughly.

 

INTRODUCTION SECTION:

Inadequacy of clear rationale and justification for choosing Chlorella  sp. As experimental organism. The manuscript mentions Chlorella  sp. As a microalga capable of high lipid accumulation. However, the rationale for selecting a specific Chlorella  strain isolated from a brackish water environment in Thailand is not clearly articulated. The manuscript should elaborate on any advantageous or distinctive features of this particular strain compared to previously studied strains. The absence of this justification weakens the reasoning for strain selection.

Needs revision: authors need to describe more, for example, any increased growth performance, original content of lipids, or exceptionally good tolerance features of the selected strain of Chlorella .

Lack of balance and consistency in reporting nutrient limitation factors (n and p). In the introduction (p. 2), nutrient limitation (N and P) and its impact on lipid building is mentioned, but they discuss further on nitrogen deprivation, while the study research is primarily on phosphorus limitation. This imposes an uneven balance between the research objectives and the problem background.

For an example, consider the sentence below:

"as suggested above, n deprivation significantly increases the lipid content but negatively affects the biomass formation of the Chlorella  sp. Cells (Praveenkumar et al., 2012). P limitation, less studied than n, might increase lipid content and unsaturated fatty acids."

The discussion is not well-structured in explaining why phosphorus limitation, rather than nitrogen, is the central focus of the study.

Required revision: the writers must restructure the paragraph to downplay nitrogen deprivation and foreground the reasoning for prioritizing phosphorus limitation in better alignment with the purpose of the study.

Erroneous or flimsy literature citations. On page 2 of the introduction, the paper says:

"higher content of lipid and unsaturated fatty acids under lower concentration of phosphate has been reported in some microalgae like Chlorella  sp. (Islam et al., 2013)."

This reference is not valid because islam et al. (2013) is concerned with general fatty acid compositions for species selection for biodiesel production, rather than phosphate-unique impacts on Chlorella  sp.

Required revision: substitute or complement this citation for more relevant research that involves direct responses of phosphorus impacts on Chlorella  sp., for instance:

• Feng et al., 2012
• Liang et al., 2013
• Sharma et al., 2012

Insufficient discussion on the role of heavy metals (Fe, Co, Pb). While the paper is studying the impact of heavy metals on microalgae, there is no extensive elaboration of how the metals affect lipid biosynthesis. There is only a general statement given by the paper:

"several studies have reported the oxidative stress due to heavy metals especially fe and co on microalgae leading to the increase of lipid content (Liu et al., 2008, Battah et al., 2015, Carfagna et al., 2013, Islam et al., 2013)."

The introduction should briefly mention the specific enzymatic pathways or cellular systems affected by these metals that trigger oxidative stress and promote lipid accumulation.

Required revision: add a more specific explanation of how Fe, Co, and Pb are involved in lipid biosynthesis, the mechanisms of oxidative stress they induce, or at least clarify why these metals were selected, citing more appropriate and targeted references.

Lack of a clear research objective and scientific hypothesis. The introduction does not clearly articulate the research aim or hypothesis. Currently, the study objective is only briefly mentioned in the abstract and not reiterated or detailed at the end of the introduction. This reduces the coherence and professionalism of the introductory section.

Required revision: state the research objective and scientific hypothesis expressly as part of an introduction conclusion paragraph, for example:

"therefore, the current study specially aims to explore (1)… (2)… under the hypothesis that…."

MATERIALS AND METHODS

1. Lack of specific information on the algal strain used. The manuscript mentions only “Chlorella sp. Isolated from natural brackish water in Thailand,” but provides no detailed classification, such as strain code, depository, or original isolation method.

Required revision: provide additional information on the collection locality (e.g., GPS coordinates), conditions of the environment at the isolation site, and where available, genetic characterization of the strain based on 18s rDNA or its sequencing.

2. Absence of definitive rationale for selected heavy metal levels (Fe, Co, Pb). It uses Fe (6 µm), Co (17 mm), and Pb (10 µm) but does not provide a justification for these particular concentrations. Specifically, the cobalt concentration is rather high as compared to typical ranges and needs more explanation.

Required revision: justify the scientific basis for choosing each metal concentration more explicitly, referencing literature or pilot data.

3. Absence of initial lipid quality verification. The authors do not indicate any method for evaluating the quality of extracted lipids (e.g., purity, contaminants, potential for oxidation), which would impact the validity of the fame results.

Required revision: describe if initial screenings were conducted on the lipid extracts prior to fame conversion, for instance, tlc or gc to confirm purity and reduce interference from other compounds such as carotenoids or chlorophyll.

4. Absence of details regarding the sod and cat enzyme assay technique. It reports that the activities of the enzymes were assessed by a commercial kit (Sigma Aldrich, USA), but does not give more information (e.g., product code, steps of the protocol, sample pretreatment).

Required revision: describe detailed information for the enzyme assay kits (product ID, concentration of sample, detailed protocol), and succinctly define the measurement principle (e.g., wavelength of absorbance, substrate employed).

5. Unclear methodology for calculating tece (total energy conversion efficiency). The formula for calculating input light energy (18.88 Kj) and the heating value of lipid (36.3 Kj/g) is not clearly explained, which may reduce transparency.

Required revision: provide details on how input light energy was calculated (e.g., flask surface area, illumination time, light intensity), and cite the source for the lipid heating value.

6. Inadequate discussion of statistical assumption testing. Even though the paper reports conducting t-tests and graphpad, it does not explain how statistical assumptions were confirmed (e.g., normality by Shapiro-Wilk test, homogeneity of variance by Levene’s or Bartlett’s test).

Required revision: it must also include the assumption tests that have been performed pre-t-tests to increase statistical rigor of findings.

SECTION 3.1

Lack of experimental data for the lipid composition

The authors made a significant claim:

"it is worth mentioning that under p deprivation, there was an alteration of the lipid composition, e.g. Diacylglyceryl-trimethylhomoserine (DGTS), diacylglyceryl-hydroxymethyltrimethyl-β-alanine (DGTA) and diacylglyceryl-carboxyhydroxy-methylcholine (DGCC)."

However, the statement is supported only by references (Sharma et al., 2012; Kalisch et al., 2016; Khozin-goldberg & Cohen, 2006), but not by experimental evidence from the current study. This reduces the impact of the discovery.

Required revision: the authors should include their own experimental data regarding specific lipid changes under phosphorus deprivation, such as HPLC, GC-MS, or LC-MS analyses of phospholipids and glycolipids under p-limited conditions.

Unclear explanation of the effect of p deprivation on biomass and lipid yield

The paper contends:

"as expected, the biomass did not increase under p deprivation at day 7 (fig. 1a) resulting in the lower lipid production than the control with 100% p (fig. 1c)."

However, this is insufficient in mechanistic depth. Why was it "expected" that p deprivation would reduce biomass and, in turn, lipid yield? The explanation lacks academic rigor.

Need for revision: this section needs greater explanation of what happens to the metabolic processes due to limitation of phosphorus (e.g., suppressing ATP, NADPH, ribosome, nucleic acid formation), thereby repressing cell division as well as biomass formation, and thus total lipid production.

Intermediate levels of phosphorus: missing data

Section 3.1 compares only two broad conditions (0% p and 100% p). However, the title refers to "phosphorus limitation," which implies the need for several intermediate phosphorus concentrations to assess nuanced effects, not just complete depletion.

Required revision: include results from additional conditions (e.g., 25%, 50%, 75% of optimal phosphorus levels) to provide clearer insight into the relationship between phosphorus availability and lipid accumulation.

Absence of physiological and stress-related data under phosphorus limitation

While the authors implicate phosphorus limitation as an explanation for decreased chlorophyll and biomass, they present no detailed values of chlorophyll content or of physiological variables like photosynthesis efficiency (e.g., Fv/Fm) or growth rate.

Required revision: provide direct measurements of physiological impacts, such as chlorophyll content, photosynthetic performance using pam fluorometry, or cell growth rate assessed via cell counting. These will support the interpretations more robustly.

Generalized and underdeveloped interpretations

The paper says:

"therefore, various strains of Chlorella  responded differently to p deprivation for the production of lipids."

This is a general observation and does not add greatly to the scholarly literature. It is more helpful to attempt an explanation for why these disparities exist.

Required revision: expand on this by discussing underlying physiological, biochemical, or genetic mechanisms (e.g., differences in gene expression related to phosphorus metabolism or lipid synthesis) to explain strain-specific responses to phosphorus limitation.

SECTION 3.2

1. Repetition and lack of consistency of presentation

There is conspicuous redundancy here, namely for the impact of Fe³⁺, which is mentioned both initially and finally in the section:

"Fe³⁺ was used to increase the lipid content in microalgae (Das et al., 2019)."

Required revision: eliminate or condense redundant material to ensure clarity, coherence, and an appropriate professional tone for reporting the results.

2. Absence of rationale for utilizing very high cobalt concentration (17 mm)

They incubated cells in 17 mm Co²⁺, several orders of magnitude above typically reported concentrations for analogous work (in the several µm range), but they fail to explain themselves for choosing to do so or to properly address the potential toxicity of that concentration:

"in the present study, 17 mm cobalt nitrate (10 times of normal bg11)."

Required revision: state an explicit rationale for selecting such a high co²⁺ concentration based on literature or preliminary results, and include more detailed consideration of toxicity risk for such a concentration.

3. Lack of mechanistic explanation for the differential Chlorella sp. Response to Fe³⁺ and Co²⁺

The authors describe the observed phenomena:

"with p deprivation, adding Co²⁺ did not raise the biomass, but raised lipid content…, but adding Fe³⁺ barely affected the lipid content."

Nonetheless, no mechanistic rationale is presented for why there is an apparent difference between the consequence of these metals, or why Co²⁺ quickly stimulates the accumulation of lipids but Fe³⁺ does not.

Required revision: provide more elaborate discussions of possible mechanisms, such as metal uptake variations, effects on photosynthetic apparatus (e.g., PSII), oxidative profile of stresses, lipid metabolism enzymes, or gene expression patterns.

4. Lack of supplemental data on oxidative stress indicators under Fe³⁺ and Co²⁺ treatments

The manuscript makes general claims that metal exposure induces stress and stimulates lipid storage but reports no direct values for markers of oxidative stress (e.g., ROS, MDA, H₂O₂) or antioxidant enzymes (SOD, CAT) under these metal exposure conditions. This is in contrast to later sections (e.g., 3.3), where relevant values are reported for Pb.

Required revision: include oxidative stress values (ROS levels, MDA, SOD, CAT) for Fe³⁺ and Co²⁺ treatment to justify the association between oxidative stress and formation of lipids under these conditions.

5. Absence of dose-response conditions for concentrations of metals

Only one concentration of each metal (Fe³⁺: 6 µm, Co²⁺: 17 mm) was assessed. This restricts the capacity to discern optimum or threshold effects of each metal.

Needs revision: include 2–3 levels of concentration for each metal to allow for better assessment of the effective dosage range and for determining the most appropriate conditions for maximizing the lipid content.

SECTION 3.3

1. Lack of clear justification for the selected Pb²⁺ concentration (10 µm) and tested range

They employed 10 µm Pb²⁺, but did not explicitly justify the scientific basis for the use of that particular concentration. While they reported other literature (Piotrowska-niczyporuk 2015, Bajguz 2011), they did not give an extensive justification for the use of that particular level.

Required revision: provide a better scientific justification for treating with 10 µm Pb²⁺, and include more concentrations to more accurately define thresholds of toxicity and of optimality.

2. Inconsistencies between analysis and interpretation of lipid data

The authors state:

"the lipid content was slightly increased under 100% p added with Pb²⁺ compared to the control without Pb²⁺. However, Pb²⁺ was unable to increase lipid content under 50 and 0% p."

Nonetheless, that interpretation seems superficial and somewhat incongruent, as elsewhere in the paper (further along section 3.3), the authors assert that lipid production had increased at 50% p with Pb²⁺ (figure 3f).

Required revision: clarify the difference between the content of lipids (% lipid per biomass) and lipid production (g lipid/l), and explain why Pb²⁺ had different impacts upon these two variables below 50% p to avoid confusion and contradiction.

3. Lack of information regarding long-term toxicity and Pb accumulation within cells

Only short-term effects (7 days) are examined in the study. It is relevant for environmental safety and applications but does not cover the possible long-term toxicity and accumulation of Pb in algal cells.

Required revision: add Pb accumulation biomass measurements (e.g., ICP-MS) and address the commercial biodiesel production or feedstock use implications due to potential heavy metal accumulation.

4. Lack of adequate analysis of the connection between antioxidant enzymes (SOD, CAT) and lipid accumulation mechanisms

They only give descriptive statements:

"Pb treatment enhanced ros, and cells were sufficiently safeguarded against ROS through sod and cat enzymes. Electron flow is towards lipid formation to counteract ROS."

However, no specific biochemical or molecular pathway is offered to explain how ros influence lipid metabolism (e.g., which lipid synthesis pathways are activated, or the role of regulatory genes like ACCASE, DGAT, PDAT).

Required revision: extend the discussion of antioxidant enzyme regulation of lipid biosynthesis on the molecular level, or, at minimum, explain what is potentially activated of the lipid metabolism processes due to oxidative stress that is elicited by Pb²⁺.

5. Missing antioxidant enzyme controls across different Pb²⁺ and phosphorus levels

Antioxidant enzyme results are presented for just one concentration (10 µm Pb²⁺, figure 4), without controls for lower or higher Pb concentrations to examine dose-dependence.

Required revision: include oxidative stress measurements (H₂O₂, MDA, SOD, CAT) at lower Pb²⁺ concentrations (e.g., 1 µm, 5 µm) to better establish a dose-response relationship.

SECTION 3.4: fatty acid profiles and biodiesel properties under Pb stress conditions

1. Lack of statistical analysis on fatty acid and biodiesel property data

Table 1 presents the fatty acid composition and biodiesel properties but lacks statistical detail (e.g., standard deviation, significance testing, or comparative analysis across conditions).

Required revision: provide full statistical details (mean ± SD, anova or equivalent tests) to determine the reliability and significance of noted differences.

2. Lack of control data on Pb content of biomass and lipid samples

No Pb accumulation data are given for algal biomass or extracted oil by the authors, although there would be possibilities for compromising the quality and safety of the resultant biodiesel.

Required revision: add ICP-MS or similar analyses to measure Pb accumulation in algal biomass and extracted oil.

3. Insufficient mechanistic explanation for changes in specific fatty acids

The manuscript only describes the shifts in fatty acid levels (e.g., C16:0, C18:0, C16:1) without explaining why phosphorus limitation and Pb²⁺ exposure cause these changes.

Required revision: provide mechanistic insights or biochemical reasoning—such as the impact of Pb²⁺ and phosphorus limitation on fatty acid synthesis enzymes (e.g., accase, fas, dgat) and related metabolic pathways that influence the SFA/UFA ratio.

4. No analysis of oxidative stability and storage capacity of biodiesel

Oxidative stability is an important consideration for evaluating the viability of biodiesel, but the authors did not include any such measure.

Recommended revision: add oxidative stability tests (e.g., Rancimat test) for determining the storage performance and practical usability of the resulting biodiesel.

5. Lacking clear discussion of biodiesel parameters

The paper just presents these biodiesel values without critically evaluating the effect of Pb and phosphorus on each of these values.

Required revision: discuss detailedly why certain conditions (p levels, Pb²⁺) have an impact on single parameters, supplemented by explicit references to alterations of fatty acid composition.

6. Lack of long-term assessment of biodiesel quality

All data reflect only short-term measurements (e.g., 24 hours), without addressing how biodiesel quality may change over time.

Required revision: perform further experiments to track quality of biodiesel for longer time intervals (at least 2–4 weeks) to assess storage stability and practical usability.

CONCLUSION

1. Conclusion does not adequately reflect key limitations of the study

The conclusion emphasizes the strengths of the study but entirely overlooks its limitations, such as short experimental duration and the absence of analysis on heavy metal accumulation.

Necessary update: insert an independent paragraph explicitly covering main shortcomings, such as long-term metal toxicity, absence of molecular mechanism study, and limited scalability.

2. Lack of explicit information on optimum experimental conditions

Most conclusions mostly talk about stress conditions but do not explicitly define the best combination (e.g., metal and phosphorus concentrations) for optimizing anabolic content as well as for better-quality biodiesel.

Required revision: specify the optimal condition based on experimental data.

3. Lack of concrete plans for future work

No clear guidance is presented in the conclusion for future work overcoming existing limitations or extending results.

Needs revision: incorporate specific research recommendations such as studying molecular mechanisms, assessing long-term toxicity, or conducting pilot-scale production tests.

4. No definitive statement on the practical value for industrial and environmentally oriented applications

It discusses general advantages of microalgae for the production of biodiesel but does not present the unique advantages of the present study over the existing literature.

Required revision: clearly emphasize practical value—such as potential for wastewater treatment with metal removal, economic or environmental benefits relative to other feedstocks.

5. No discussion of risks and challenges in real-world application

Summary is not accompanied by an assessment of risks (e.g., toxicity or contamination of heavy metals) and technical challenges associated with commercial use.

Required revision: add a note in the conclusion discussing potential risks and critical considerations before scaling up the application for industrial use.

Author Response

1. Scientific and practical strengths:

Topic selection is very relevant, involving the production of biodiesel from microalgae, specifically Chlorella  sp., and is consistent with present day trends of renewable energy production and greenhouse gas reduction. This research presents an integrated effect of phosphorus limitation and metal stress, which is less addressed in the literature, providing insights into the metabolism of lipids and tolerance of stress of microalgae.

It is deliberately set up as an experiment that integrates nutrient constraint (phosphorus) into toxic metal treatment (Fe, Co, Pb). It provides detailed physiological outcomes, supporting the validity and significance of outcomes for the research community.

An important aspect is the characterization of lipids and biodiesel on several dimensions. Whereas the paper does measure the content of the lipids, it proceeds to analyze fatty acid composition, and for those relevant biodiesel properties, makes conscientious comparison to astm and en standards. This further enhances the work's applied relevance.

Importantly, the authors also look at markers of oxidative stress (H₂O₂, MDA) as well as protective enzymatic activities (SOD, CAT), adding richness to physiological understanding and suggestive of a possible mechanism whereby Pb stress could impact lipid metabolism.

Response: Thanks for constructive comments on the manuscript.

2. Key constraints to be met for Q1 preparedness:

-A key limitation is the lack of molecular-level mechanistic analysis. The explanations for lipid accumulation remain observational, with no detailed exploration of specific lipid metabolic pathways or regulatory genes (e.g., DGAT, PDAT). In the context of current research, molecular insights are essential for elevating the manuscript’s scientific merit.

Response: Thanks for the comments to improve the merit of the manuscript. We very much appreciate this insightful suggestion. The detailed molecular study as suggested by the reviewer will be done in future study to extensively elucidate the mechanism underlying the results observed in the present manuscript.

-Novelty is also quite incremental. As the study presents heavy metal stress to the standard nutrient deprivation model, that pairing, while valid, lacks the disruptive impact typically desired for Q1 articles. What is presented is not significantly different from existing knowledge or technique.

Response: We agree with the reviewer that the novel aspect is incremental. However, the new  approach of combining the effects of two different stress conditions simultaneously on lipids content in microalgae has not been reported.  Therefore, this study establishes the platform for further in-depth investigation, particularly in the context of improving lipids yield for biodiesel production.

-Not enough has been discussed regarding the long-term toxicity and accumulation potential of heavy metals. Whereas short-term enhancement of lipids is illustrated, there is no adequate consideration of the possible ecological as well as commercial consequences of residual heavy metals in biomass.

Response: Thanks for the comments. It is unfortunate that we did not measure how much the metal, particularly Co which increased lipid content, accumulated inside the cells. However, the presence of low concentration of Co at 17 µM under 0 % P did not affect cell survival despite its growth inhibition as shown in Fig. 2A. Therefore, the consequences of residual heavy metals in biomass should be minimal.  The long-term toxicity on the cells and accumulation potential of heavy metals were discussed in the text (Lines 248-254).

-It only studied one strain of Chlorella , limiting generalizability. It is unknown, without cross-strain comparison, whether reported effects are strain-specific or universally true for other species of microalgae.

Response: Thanks for invaluable comments. Similar study in other species of microalgae will be further investigated to reach the conclusion regarding the effects of combined stress conditions on lipids content. However, discussion on this issue has been provided (Lines 160-169).

3.. Recommendations for improvement: in particular:

In order to attain Q1 quality, authors are recommended to:

• Additional molecular characterization, such as rt-qpcr, transcriptomics, proteomics, or key enzyme assays (e.g., DGAT, PDAT, ACCASE), can be integrated to present an explanation of cellular processes of lipid accumulation.
• To quantify the heavy metal loading of algal cells and determine the toxicity and environmental effects of the post-treatment biomass, thereby delineating risks from benefits.
• Expand the study to include multiple Chlorella strains or related microalgae to improve generalizability.
• Provide long-term data to assess continued lipid productivity under longer-lasting pressure conditions, providing an indicator of scalability for pilot or commercial uses.

Response: All these recommendations will be taken into our next plan of research to deepen the well defined mechanistic aspects regarding the effects of P-deprived and metal stress conditions on changes in lipids content. Thank you for the precious and insightful recommendations for planning Q1 quality of future work intended for Phycology.

1. General conclusion:

The paper shows an adequate course with good experimental results and good practical significance. Nevertheless, as of now, it does not exhibit mechanistic profundity or unique innovation typical of leading-edge journals. It is adequate for mid-tier journals Q2–Q3, but for consideration for high-impact Q1 journals, there is a need for the authors to include extensive molecular and mechanistic investigations, expand comparative datasets, and cover heavy metal accumulation hazard thoroughly.

Response: We do agree with the reviewer. We will endeavor to incorporate all recommendations of the reviewer in our future manuscript as an extension of the present study to attain the quality of another Q1 article for Phycology.

 

INTRODUCTION SECTION:

Needs revision: authors need to describe more, for example, any increased growth performance, original content of lipids, or exceptionally good tolerance features of the selected strain of Chlorella .

Response: The revision as suggested has been done (Lines 86-88).

For an example, consider the sentence below:

"as suggested above, n deprivation significantly increases the lipid content but negatively affects the biomass formation of the Chlorella  sp. Cells (Praveenkumar et al., 2012). P limitation, less studied than n, might increase lipid content and unsaturated fatty acids."

The discussion is not well-structured in explaining why phosphorus limitation, rather than nitrogen, is the central focus of the study.

Required revision: the writers must restructure the paragraph to downplay nitrogen deprivation and foreground the reasoning for prioritizing phosphorus limitation in better alignment with the purpose of the study.

Response: The suggested sentences have been incorporated into the text . Also the reasoning for prioritizing phosphorus limitation with the added benefit has been described (Lines 52-61).

Erroneous or flimsy literature citations. On page 2 of the introduction, the paper says:

"higher content of lipid and unsaturated fatty acids under lower concentration of phosphate has been reported in some microalgae like Chlorella  sp. (Islam et al., 2013)."

This reference is not valid because islam et al. (2013) is concerned with general fatty acid compositions for species selection for biodiesel production, rather than phosphate-unique impacts on Chlorella  sp.

Required revision: substitute or complement this citation for more relevant research that involves direct responses of phosphorus impacts on Chlorella  sp., for instance:

• Feng et al., 2012
• Liang et al., 2013
• Sharma et al., 2012

Response: "higher content of lipid and unsaturated fatty acids under lower concentration of phosphate has been reported in some microalgae like Chlorella  sp. (Islam et al., 2013)." This sentence has been removed (Lines 51-52).

Insufficient discussion on the role of heavy metals (Fe, Co, Pb). While the paper is studying the impact of heavy metals on microalgae, there is no extensive elaboration of how the metals affect lipid biosynthesis. There is only a general statement given by the paper:

"several studies have reported the oxidative stress due to heavy metals especially fe and co on microalgae leading to the increase of lipid content (Liu et al., 2008, Battah et al., 2015, Carfagna et al., 2013, Islam et al., 2013)."

The introduction should briefly mention the specific enzymatic pathways or cellular systems affected by these metals that trigger oxidative stress and promote lipid accumulation.

Required revision: add a more specific explanation of how Fe, Co, and Pb are involved in lipid biosynthesis, the mechanisms of oxidative stress they induce, or at least clarify why these metals were selected, citing more appropriate and targeted references.

Response: Thanks for the comments. However, the main purpose of this study is to test the effect of various metals on the lipid content and to use the increased lipids for biodiesel production. It is too much detail to describe the mechanisms involved which might distract the interest of potential readers in the field. However, some description on the suggested comments have been provided (Lines 67-73).

 

Lack of a clear research objective and scientific hypothesis. The introduction does not clearly articulate the research aim or hypothesis. Currently, the study objective is only briefly mentioned in the abstract and not reiterated or detailed at the end of the introduction. This reduces the coherence and professionalism of the introductory section.

Required revision: state the research objective and scientific hypothesis expressly as part of an introduction conclusion paragraph, for example:

"therefore, the current study specially aims to explore (1)… (2)… under the hypothesis that…."

Response: We have added the suggested sentence to the last part of Introduction section (Lines 73-76).

MATERIALS AND METHODS

1. Lack of specific information on the algal strain used. The manuscript mentions only “Chlorella sp. Isolated from natural brackish water in Thailand,” but provides no detailed classification, such as strain code, depository, or original isolation method.

Required revision: provide additional information on the collection locality (e.g., GPS coordinates), conditions of the environment at the isolation site, and where available, genetic characterization of the strain based on 18s rDNA or its sequencing.

     2. Absence of definitive rationale for selected heavy metal levels (Fe, Co, Pb). It uses Fe (6 µm), Co (17 mm), and Pb (10 µm) but does not provide a justification for these particular concentrations. Specifically, the cobalt concentration is rather high as compared to typical ranges and needs more explanation.

Required revision: justify the scientific basis for choosing each metal concentration more explicitly, referencing literature or pilot data.

     3. Absence of initial lipid quality verification. The authors do not indicate any method for evaluating the quality of extracted lipids (e.g., purity, contaminants, potential for oxidation), which would impact the validity of the fame results.

Required revision: describe if initial screenings were conducted on the lipid extracts prior to fame conversion, for instance, tlc or gc to confirm purity and reduce interference from other compounds such as carotenoids or chlorophyll.

      4. Absence of details regarding the sod and cat enzyme assay technique. It reports that the activities of the enzymes were assessed by a commercial kit (Sigma Aldrich, USA), but does not give more information (e.g., product code, steps of the protocol, sample pretreatment).

Required revision: describe detailed information for the enzyme assay kits (product ID, concentration of sample, detailed protocol), and succinctly define the measurement principle (e.g., wavelength of absorbance, substrate employed).

      5. Unclear methodology for calculating tece (total energy conversion efficiency). The formula for calculating input light energy (18.88 Kj) and the heating value of lipid (36.3 Kj/g) is not clearly explained, which may reduce transparency.

Required revision: provide details on how input light energy was calculated (e.g., flask surface area, illumination time, light intensity), and cite the source for the lipid heating value.

      6. Inadequate discussion of statistical assumption testing. Even though the paper reports conducting t-tests and graphpad, it does not explain how statistical assumptions were confirmed (e.g., normality by Shapiro-Wilk test, homogeneity of variance by Levene’s or Bartlett’s test).

Required revision: it must also include the assumption tests that have been performed pre-t-tests to increase statistical rigor of findings.

Response: Thanks for all suggestions on M & M section. We very much appreciate the reviewer’s critiques. However, we feel that the description in all of this section has met the required standard of most scientific journals, as we have done for many years regarding research publications especially in the area of algal biotechnology (see some examples below). Nevertheless, some of the comments will be useful for our future work, and again we thank the reviewer for such a critical review.

 

Thongtha S, Kittiwongwattana C, Incharoensakdi A, Phunpruch S (2025) Light emitting diode illumination enhances biomass, pigment and lipid production in halotolerant cyanobacterium Aphanothece halophytica. Phycology 5: 12; doi.org/ 10.3390/phycology5020012

Ramprakash B, Sivaramakrishnan R, Subramani K, Incharoensakdi A (2024) Iron oxide treated Chlorella sp. for enhanced biomass and lipid content coupled to  fermentative hydrogen production by Enterobacter aerogenes using hydrolyzate from pretreated biomass. Int. J. Hydrogen Energy 73: 43-53; doi.org/10.1016/j.ijhydene.2024.06.003

Sivaramakrishnan R, Suresh S, Incharoensakdi A (2024) Development of Chlamydomonas sp. biorefinery for sustainable methyl ester and malic acid production. Fuel 371: 132017; doi.org/10.1016/j.fuel.2024.132017

Sivaramakrishnan R, Incharoensakdi A (2023) UV mutagenesis followed by hydrogen peroxide treatment ameliorates lipid production and omega-3 fatty acids levels in Chlorella sp.  Algal Res. 74: 103195; doi.org/10.1016/j.algal.2023.103195

Sivaramakrishnan R, Incharoensakdi A (2017) Enhancement of total lipid yield by nitrogen, carbon, and iron supplementation in isolated microalgae. J. Phycol. 53: 855-868.

 

 

SECTION 3.1

Unclear explanation of the effect of p deprivation on biomass and lipid yield.

Need for revision: this section needs greater explanation of what happens to the metabolic processes due to limitation of phosphorus (e.g., suppressing ATP, NADPH, ribosome, nucleic acid formation), thereby repressing cell division as well as biomass formation, and thus total lipid production.

Response: We have added more discussion on this issue (Lines 170-174).

Intermediate levels of phosphorus: missing data

Section 3.1 compares only two broad conditions (0% p and 100% p). However, the title refers to "phosphorus limitation," which implies the need for several intermediate phosphorus concentrations to assess nuanced effects, not just complete depletion.

Required revision: include results from additional conditions (e.g., 25%, 50%, 75% of optimal phosphorus levels) to provide clearer insight into the relationship between phosphorus availability and lipid accumulation.

Response: Sorry for the misunderstanding. The title of section 3.1 is misleading, the “limitation” has been changed to “deprivation” (Line 152). The aim of this section is to test the effect of P-deprivation.

Absence of physiological and stress-related data under phosphorus limitation

While the authors implicate phosphorus limitation as an explanation for decreased chlorophyll and biomass, they present no detailed values of chlorophyll content or of physiological variables like photosynthesis efficiency (e.g., Fv/Fm) or growth rate.

Required revision: provide direct measurements of physiological impacts, such as chlorophyll content, photosynthetic performance using pam fluorometry, or cell growth rate assessed via cell counting. These will support the interpretations more robustly.

Response: The aim of this section is to test the effect of P-deprivation on the changes in biomass and lipid content. In general, chlorophyll content and photosynthetic performance are in parallel or in the same trend as the biomass, which represents cell growth. We agree with the reviewer that such measurements will allow for more robust interpretations, and these suggestions will be planned for future work.

Generalized and underdeveloped interpretations

The paper says:

"therefore, various strains of Chlorella  responded differently to p deprivation for the production of lipids."

This is a general observation and does not add greatly to the scholarly literature. It is more helpful to attempt an explanation for why these disparities exist.

Required revision: expand on this by discussing underlying physiological, biochemical, or genetic mechanisms (e.g., differences in gene expression related to phosphorus metabolism or lipid synthesis) to explain strain-specific responses to phosphorus limitation.

Response: Thanks for the comments. We discussed that the response of Chlorella to P deprivation may not be strain-specific. However, it remains to be further investigated with respect to biochemical and/or genetic mechanisms (Lines 160-165). Additional discussion is also provided (Lines 165-169).

The response of microalgae to P-deprivation is rather complicated involving a complex interplay of metabolic shifts among the synthesis and break down of various macromolecules. For example, P-deprivation caused a severe decrease in protein and a considerable increase in lipids including triacylglycerol, but had little effect on carbohydrate level and biomass production of Nannochloropsis oceanica (Shi et al., 2020).

SECTION 3.2

1. Repetition and lack of consistency of presentation

There is conspicuous redundancy here, namely for the impact of Fe³⁺, which is mentioned both initially and finally in the section:

"Fe³⁺ was used to increase the lipid content in microalgae (Das et al., 2019)."

Required revision: eliminate or condense redundant material to ensure clarity, coherence, and an appropriate professional tone for reporting the results.

Response: The repetition/redundancy part has been deleted (Lines 215-224 and 232-234).

      2. Absence of rationale for utilizing very high cobalt concentration (17 mm)

They incubated cells in 17 mm Co²⁺, several orders of magnitude above typically reported concentrations for analogous work (in the several µm range), but they fail to explain themselves for choosing to do so or to properly address the potential toxicity of that concentration:

"in the present study, 17 mm cobalt nitrate (10 times of normal bg11)."

Required revision: state an explicit rationale for selecting such a high co²⁺ concentration based on literature or preliminary results, and include more detailed consideration of toxicity risk for such a concentration.

Response: Thank you for raising this important point. It was an inadvertently made mistake. The actual 17 µM Co was used in the experiments and the corrections have been made in the text. This low concentration was in the range used by previous studies (Lines 264-269).

        3. Lack of mechanistic explanation for the differential Chlorella sp. Response to Fe³⁺ and Co²⁺

The authors describe the observed phenomena:

"with p deprivation, adding Co²⁺ did not raise the biomass, but raised lipid content…, but adding Fe³⁺ barely affected the lipid content."

Nonetheless, no mechanistic rationale is presented for why there is an apparent difference between the consequence of these metals, or why Co²⁺ quickly stimulates the accumulation of lipids but Fe³⁺ does not.

Required revision: provide more elaborate discussions of possible mechanisms, such as metal uptake variations, effects on photosynthetic apparatus (e.g., PSII), oxidative profile of stresses, lipid metabolism enzymes, or gene expression patterns.

Response: More discussion has been provided (Lines 250-254)

Microalgae utilize appropriate mechanisms to uptake various kinds of metals and further detoxify them. Different mechanisms may be used by Chlorella to deal with the presence of Fe and Co. The absorption of Fe and Co and the transport into the cells can be managed by different  transporters, and this can lead to different level of ROS generation caused by Fe and Co.

 

         4. Lack of supplemental data on oxidative stress indicators under Fe³⁺ and Co²⁺ treatments

The manuscript makes general claims that metal exposure induces stress and stimulates lipid storage but reports no direct values for markers of oxidative stress (e.g., ROS, MDA, H₂O₂) or antioxidant enzymes (SOD, CAT) under these metal exposure conditions. This is in contrast to later sections (e.g., 3.3), where relevant values are reported for Pb.

Required revision: include oxidative stress values (ROS levels, MDA, SOD, CAT) for Fe³⁺ and Co²⁺ treatment to justify the association between oxidative stress and formation of lipids under these conditions.

Response: The oxidative stress indicators under Fe³⁺ and Co²⁺ treatments were not analyzed in this study. However, it has been previously reported that Chlorella sp. showed a high tolerance under treatment with 34 µM of cobalt and cells showed elevated levels of stress biomarkers (Kashyap et al., 2021). The concentration of Co used in the reported study (34 µM) was in the same order of magnitude, which is not much different from that in our study (17µM). For Fe, The addition of iron up to 10 µM increased final cell densities by nearly 2-fold while 1 mM iron was toxic (Wan et al., 2014). Moreover, Fe treatment in our study showed no growth inhibition (Fig. 2A), thus unlikely to cause oxidative stress (Lines 274-281).

5. Absence of dose-response conditions for concentrations of metals

Only one concentration of each metal (Fe³⁺: 6 µm, Co²⁺: 17 mm) was assessed. This restricts the capacity to discern optimum or threshold effects of each metal.

Needs revision: include 2–3 levels of concentration for each metal to allow for better assessment of the effective dosage range and for determining the most appropriate conditions for maximizing the lipid content.

Response: Thanks for the comments. Our primary aim was to test the effect of various types of metal in combination with P limitation on lipids content and lipids production for application in biodiesel production. The concentrations of metals used were in similar range of those in previous studies. However, the suggested comments will be incorporated into future work.

 

SECTION 3.3

1. Lack of clear justification for the selected Pb²⁺ concentration (10 µm) and tested range

They employed 10 µm Pb²⁺, but did not explicitly justify the scientific basis for the use of that particular concentration. While they reported other literature (Piotrowska-niczyporuk 2015, Bajguz 2011), they did not give an extensive justification for the use of that particular level.

Required revision: provide a better scientific justification for treating with 10 µm Pb²⁺, and include more concentrations to more accurately define thresholds of toxicity and of optimality.

Response: The justification of using 10 µM Pb was added (Lines 285-288).

 

     2.  Inconsistencies between analysis and interpretation of lipid data

The authors state:

"the lipid content was slightly increased under 100% p added with Pb²⁺ compared to the control without Pb²⁺. However, Pb²⁺ was unable to increase lipid content under 50 and 0% p."

Nonetheless, that interpretation seems superficial and somewhat incongruent, as elsewhere in the paper (further along section 3.3), the authors assert that lipid production had increased at 50% p with Pb²⁺ (figure 3f).

Required revision: clarify the difference between the content of lipids (% lipid per biomass) and lipid production (g lipid/l), and explain why Pb²⁺ had different impacts upon these two variables below 50% p to avoid confusion and contradiction.

Response: The suggested comment with more discussion has been incorporated into the text (Lines 297-303).

      3. Lack of information regarding long-term toxicity and Pb accumulation within cells

Only short-term effects (7 days) are examined in the study. It is relevant for environmental safety and applications but does not cover the possible long-term toxicity and accumulation of Pb in algal cells.

Required revision: add Pb accumulation biomass measurements (e.g., ICP-MS) and address the commercial biodiesel production or feedstock use implications due to potential heavy metal accumulation.

Response: Thanks for the comments. Unfortunately we did not measure Pb accumulation inside the cells. It is beyond the scope of the present study. Short-term study should be suitable to increase lipid production for use in the production of biodiesel. Cells would be subjected to short term metal stress, and the toxicity that might occur would be minimized. This would allow cells to respond to the stress by adjusting their metabolism towards the synthesis of lipids and other compounds of interest.

        4. Lack of adequate analysis of the connection between antioxidant enzymes (SOD, CAT) and lipid accumulation mechanisms

They only give descriptive statements:

"Pb treatment enhanced ros, and cells were sufficiently safeguarded against ROS through sod and cat enzymes. Electron flow is towards lipid formation to counteract ROS."

However, no specific biochemical or molecular pathway is offered to explain how ros influence lipid metabolism (e.g., which lipid synthesis pathways are activated, or the role of regulatory genes like ACCASE, DGAT, PDAT).

Required revision: extend the discussion of antioxidant enzyme regulation of lipid biosynthesis on the molecular level, or, at minimum, explain what is potentially activated of the lipid metabolism processes due to oxidative stress that is elicited by Pb²⁺.

Response: Oxidative stress is  a consequence of an imbalance between the production of reactive oxygen species (ROS) and the antioxidant system which includes the function of various antioxidant enzymes. Oxidative stress regulates the expression and activity of lipid metabolism-related enzymes, leading to the increase in fatty acid synthesis. Our previous study in Chlorella sp. showed an increase of ACC activity, the enzyme for  fatty acid synthesis, as well as the up-regulation of some important genes in fatty synthesis pathway, under oxidative stress caused by UV radiation and H2O2 (Sivaramakriishnan & Incharoensakdi, 2023). The  up- and down-regulation of some genes in fatty acid synthesis pathway can lead to the change in SFA/UFA ratio. (Lines 364-372).

        5. Missing antioxidant enzyme controls across different Pb²⁺ and phosphorus levels

Antioxidant enzyme results are presented for just one concentration (10 µm Pb²⁺, figure 4), without controls for lower or higher Pb concentrations to examine dose-dependence.

Required revision: include oxidative stress measurements (H₂O₂, MDA, SOD, CAT) at lower Pb²⁺ concentrations (e.g., 1 µm, 5 µm) to better establish a dose-response relationship.

Response: Thanks for constructive suggestions. We will persue a dose-response relationship for Pb concentration as well as for different levels of P in future work. This will shed light on the mechanism of oxidative stress in Chlorella sp.

SECTION 3.4: fatty acid profiles and biodiesel properties under Pb stress conditions

1. Lack of statistical analysis on fatty acid and biodiesel property data

Table 1 presents the fatty acid composition and biodiesel properties but lacks statistical detail (e.g., standard deviation, significance testing, or comparative analysis across conditions).

Required revision: provide full statistical details (mean ± SD, anova or equivalent tests) to determine the reliability and significance of noted differences.

Response: The suggested comment was added (Lines 458-460).

The data are the average values of triplicates and the maximum standard deviation among the three values was within 5% of the mean. 

       2. Lack of control data on Pb content of biomass and lipid samples

No Pb accumulation data are given for algal biomass or extracted oil by the authors, although there would be possibilities for compromising the quality and safety of the resultant biodiesel.

Required revision: add ICP-MS or similar analyses to measure Pb accumulation in algal biomass and extracted oil.

Response: Thanks for the comments. Unfortunately we did not measure Pb accumulation inside the cells. It is beyond the scope of the present study. However, Pb accumulation is planned for the extension of the present study in future work.

      3. Insufficient mechanistic explanation for changes in specific fatty acids

The manuscript only describes the shifts in fatty acid levels (e.g., C16:0, C18:0, C16:1) without explaining why phosphorus limitation and Pb²⁺ exposure cause these changes.

Required revision: provide mechanistic insights or biochemical reasoning—such as the impact of Pb²⁺ and phosphorus limitation on fatty acid synthesis enzymes (e.g., accase, fas, dgat) and related metabolic pathways that influence the SFA/UFA ratio.

Response: Pb and P limitation impact the cells by causing the oxidative stress. See response in section 3.3 item 4.

     4. No analysis of oxidative stability and storage capacity of biodiesel

Oxidative stability is an important consideration for evaluating the viability of biodiesel, but the authors did not include any such measure.

Recommended revision: add oxidative stability tests (e.g., Rancimat test) for determining the storage performance and practical usability of the resulting biodiesel.

Response: Thanks for the comments. This issue will be parts of future work.

       5. Lacking clear discussion of biodiesel parameters

The paper just presents these biodiesel values without critically evaluating the effect of Pb and phosphorus on each of these values.

Required revision: discuss detailedly why certain conditions (p levels, Pb²⁺) have an impact on single parameters, supplemented by explicit references to alterations of fatty acid composition.

Response: Pb and P do not directly affect the values of biodiesel properties. The compositions of the obtained lipids after Pb and P treatment contribute to the quality of biodiesel. The palmitic acid is the major component (54.57 %) of Chlorella  sp. under Pb and 0 % P in this study Table 1). Palmitic acid is the suitable composition for the biodiesel production and ensures good biodiesel property (Gua et al., 2018) (Lines 447-451).

 

      6. Lack of long-term assessment of biodiesel quality

All data reflect only short-term measurements (e.g., 24 hours), without addressing how biodiesel quality may change over time.

Required revision: perform further experiments to track quality of biodiesel for longer time intervals (at least 2–4 weeks) to assess storage stability and practical usability.

Response: Thanks for insightful comment. This issue will be incorporated into our future work.

CONCLUSION

1. Conclusion does not adequately reflect key limitations of the study

The conclusion emphasizes the strengths of the study but entirely overlooks its limitations, such as short experimental duration and the absence of analysis on heavy metal accumulation.

Necessary update: insert an independent paragraph explicitly covering main shortcomings, such as long-term metal toxicity, absence of molecular mechanism study, and limited scalability.

Response: The usefulness of the data in this study requires further in-depth investigation. In particular, the long-term effect of metal toxicity on lipid production as well as the amount of metal accumulating in the cells. The molecular mechanisms regarding the effect of metals on the genes involved in fatty acid synthesis also need to be further investigated. (Lines 493-497).

      2. Lack of explicit information on optimum experimental conditions

Most conclusions mostly talk about stress conditions but do not explicitly define the best combination (e.g., metal and phosphorus concentrations) for optimizing anabolic content as well as for better-quality biodiesel.

Required revision: specify the optimal condition based on experimental data.

Response: The suggestion has been incorporated into the text (Lines 481-482 and 488-489).

      3. Lack of concrete plans for future work

No clear guidance is presented in the conclusion for future work overcoming existing limitations or extending results.

Needs revision: incorporate specific research recommendations such as studying molecular mechanisms, assessing long-term toxicity, or conducting pilot-scale production tests.

Response: The proposed future work will be the long-term effect of metal toxicity on lipid production as well as the amount of metal accumulating in the cells. The molecular mechanisms regarding the effect of metals on the genes involved in fatty acid synthesis also need to be further investigated. (Lines 493-497).

       4. No definitive statement on the practical value for industrial and environmentally oriented applications

It discusses general advantages of microalgae for the production of biodiesel but does not present the unique advantages of the present study over the existing literature.

Required revision: clearly emphasize practical value—such as potential for wastewater treatment with metal removal, economic or environmental benefits relative to other feedstocks.

Response: The study on the metal accumulation in Chlorella will be beneficial in terms of the bioremediation of metal-contaminated environment, allowing the use of the organism to treat wastewater containing heavy metals. (Lines 497-499).

       5. No discussion of risks and challenges in real-world application

Summary is not accompanied by an assessment of risks (e.g., toxicity or contamination of heavy metals) and technical challenges associated with commercial use.

Required revision: add a note in the conclusion discussing potential risks and critical considerations before scaling up the application for industrial use.

Response: This suggestion has been incorporated into the text . “Nevertheless, more extensive study with the use of the consortium of various organisms is needed in real-world application due to the presence of an array of mixed heavy metals in the wastewater” (Lines 499-501).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editorial Board of Phycology (MDPI), and the Authors,

I have carefully reviewed the revised version of the manuscript entitled “Enhancement of lipids content in Chlorella sp. under phosphorus limitation and heavy metal addition for biodiesel production,” along with the detailed responses provided by the authors.

First, let me express my sincere appreciation to the authors for their substantial efforts in addressing several key issues raised in my previous review. In particular, the correction regarding the cobalt concentration (from the originally high 17 mM to an appropriate 17 µM), and clarifications provided on the selection rationale for heavy metals (Fe, Co, Pb), significantly improve the manuscript’s scientific credibility. Additionally, the authors have offered improved discussions regarding oxidative stress mechanisms and their relationship to lipid metabolism under metal-stress conditions, which enriches the overall context of the work.

Nevertheless, despite these commendable improvements, certain critical scientific issues still remain unresolved, precluding immediate acceptance of this manuscript in its current form, especially given the rigorous standards of a high-impact Q1 journal such as Phycology. I will briefly restate the most important unresolved points:

First and foremost, the manuscript still lacks critical quantitative data concerning heavy metal accumulation (Pb, Fe, Co) within the microalgal biomass. From my experience reviewing numerous manuscripts in this area, data such as ICP-MS measurements are vital for assessing the practical feasibility, environmental safety, and broader applicability of using microalgae for biodiesel production. Without such measurements, it remains difficult to adequately evaluate the potential risks and benefits of the proposed method.

Secondly, experimental data concerning oxidative stability of the produced biodiesel remains absent. Given current trends and expectations for biodiesel research, oxidative stability (e.g., measured via Rancimat analysis) is essential for determining the practical usability and commercial viability of the biodiesel product.

Thirdly, although the authors have openly acknowledged the importance of molecular-level mechanisms (such as gene expression assays, enzyme activities of key lipid biosynthesis enzymes like DGAT, PDAT, or ACCase), such critical experimental evidence has yet to be presented in the manuscript. While I greatly appreciate the authors’ intention and future commitments, these experiments represent fundamental and essential components for validating the hypothesized metabolic mechanisms outlined in the manuscript.

Moreover, statistical presentation of fatty acid composition and biodiesel properties remains overly generalized. Simply stating that the standard deviation falls within 5% of the mean does not provide sufficient transparency or statistical rigor. Clearer, more explicit statistical analyses (including individual standard deviations, statistical significance via ANOVA, or similar methods) should be provided to enhance the robustness and credibility of these findings.

Finally, it would be highly beneficial for the authors to explicitly identify and discuss the optimal conditions (particularly concentrations of phosphorus and metals) based on the current experimental data. Clearly outlining optimal conditions enhances the practical relevance and applicability of this study to potential users and researchers in the field.

Given these significant remaining issues, I conclude that the manuscript requires another round of major revision. I fully recognize the practical challenges associated with additional experiments; however, these revisions remain essential for publication in a top-tier journal like Phycology.

I genuinely appreciate the authors’ responsiveness and collaborative approach, as well as their clearly expressed commitment to addressing these critical gaps in future studies. I remain confident that incorporating the suggested improvements will significantly enhance the manuscript’s scientific quality, depth, and practical impact.

Best wishes,

Author Response

Responses to reviewer (2^nd round revision)

First, let me express my sincere appreciation to the authors for their substantial efforts in addressing several key issues raised in my previous review. In particular, the correction regarding the cobalt concentration (from the originally high 17 mM to an appropriate 17 µM), and clarifications provided on the selection rationale for heavy metals (Fe, Co, Pb), significantly improve the manuscript’s scientific credibility. Additionally, the authors have offered improved discussions regarding oxidative stress mechanisms and their relationship to lipid metabolism under metal-stress conditions, which enriches the overall context of the work.

Response: Thanks for the encouraging comments.

Nevertheless, despite these commendable improvements, certain critical scientific issues still remain unresolved, precluding immediate acceptance of this manuscript in its current form, especially given the rigorous standards of a high-impact Q1 journal such as Phycology. I will briefly restate the most important unresolved points:

Response: We are sorry that we cannot resolve all the issues raised by the reviewer. We have to apologize to the journal for submitting the manuscript that does not meet the high standard of Phycology.

First and foremost, the manuscript still lacks critical quantitative data concerning heavy metal accumulation (Pb, Fe, Co) within the microalgal biomass. From my experience reviewing numerous manuscripts in this area, data such as ICP-MS measurements are vital for assessing the practical feasibility, environmental safety, and broader applicability of using microalgae for biodiesel production. Without such measurements, it remains difficult to adequately evaluate the potential risks and benefits of the proposed method.

Response: Thanks for the critical comments. We do agree with the reviewer. However, to resolve this issue, it needs more time/expenditure/man power to obtain the data. We propose to include this issue in our next manuscript as an extension of the present manuscript.

Secondly, experimental data concerning oxidative stability of the produced biodiesel remains absent. Given current trends and expectations for biodiesel research, oxidative stability (e.g., measured via Rancimat analysis) is essential for determining the practical usability and commercial viability of the biodiesel product.

Response: Thanks for the critical comments. We do agree with the reviewer. However, to resolve this issue, it needs more time/expenditure/manpower to obtain the data. We propose to include this issue in our next manuscript as an extension of the present manuscript.

Thirdly, although the authors have openly acknowledged the importance of molecular-level mechanisms (such as gene expression assays, enzyme activities of key lipid biosynthesis enzymes like DGAT, PDAT, or ACCase), such critical experimental evidence has yet to be presented in the manuscript. While I greatly appreciate the authors’ intention and future commitments, these experiments represent fundamental and essential components for validating the hypothesized metabolic mechanisms outlined in the manuscript.

Response: Thanks for the critical comments. We do agree with the reviewer. However, to resolve this issue, it needs more time/expenditure/manpower to obtain the data. We propose to include this issue in our next manuscript as an extension of the present manuscript.

Moreover, statistical presentation of fatty acid composition and biodiesel properties remains overly generalized. Simply stating that the standard deviation falls within 5% of the mean does not provide sufficient transparency or statistical rigor. Clearer, more explicit statistical analyses (including individual standard deviations, statistical significance via ANOVA, or similar methods) should be provided to enhance the robustness and credibility of these findings.

Response: Individual standard deviations have been incorporated into the data.

Finally, it would be highly beneficial for the authors to explicitly identify and discuss the optimal conditions (particularly concentrations of phosphorus and metals) based on the current experimental data. Clearly outlining optimal conditions enhances the practical relevance and applicability of this study to potential users and researchers in the field.

Response: In the Conclusions section, we have mentioned important findings regarding lipids content and lipids production with respect to the presence/absence of P and addition of metals to facilitate other researchers to extend their work based on data from the present study.

Given these significant remaining issues, I conclude that the manuscript requires another round of major revision. I fully recognize the practical challenges associated with additional experiments; however, these revisions remain essential for publication in a top-tier journal like Phycology.

Response: Thanks for the critical comments. However, at present we are not ready to perform extra experiments since these will take time and money as well as more manpower. Our research fund is almost exhausted, and we hope to get more funding starting from October, 2025. Additionally, we have stated in the final part of the Conclusions section regarding the usefulness of the data in the present manuscript as well as the work needed to be further investigated in future studies.

I genuinely appreciate the authors’ responsiveness and collaborative approach, as well as their clearly expressed commitment to addressing these critical gaps in future studies. I remain confident that incorporating the suggested improvements will significantly enhance the manuscript’s scientific quality, depth, and practical impact.

Response: We agree with the reviewer. We very much appreciate these constructive and valuable suggestions for the betterment of science. Again thank you very much.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your thorough revisions and detailed responses to my previous review comments. I appreciate the efforts you have made in clarifying methodological details, improving the statistical presentation, and refining the discussion on the physiological responses of Chlorella sp. to combined phosphorus limitation and heavy metal stress.

However, several key scientific issues that were identified in earlier review rounds remain unresolved in the current manuscript. In particular, the absence of experimental data on heavy metal accumulation (Pb, Fe, Co), oxidative stability measurements for the produced biodiesel, and molecular-level evidence (gene expression or enzyme assays such as DGAT, PDAT, ACCase) continues to limit the depth, novelty, and applied relevance of the study. I fully understand the constraints in resources and time that you have outlined in your responses. Nevertheless, these components are considered essential for a Q1-level publication in Phycology.

Given the current scope and limitations of the data, the manuscript still does not fully meet the journal’s high standards for scientific rigor and completeness. I will forward my full assessment to the Editor for final decision-making.

I appreciate your constructive engagement throughout the review process and hope that my comments will be useful should you wish to further develop this work in the future.

Sincerely,