Productivity and Carbon Utilization of Three Green Microalgae Strains with High Biotechnological Potential Cultivated in Flat-Panel Photobioreactors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript of reference 3756717 entitled “Productivity and Carbon Utilization of Three Green Microalgae Strains with High Biotechnological Potential Cultivated in Flat-Panel Photobioreactors”, submitted to the journal Phycology (MDPI) by Gabrielyan et al., is an original, unpublished study that evaluates the biotechnological possibilities of three green microalgae strains (Neochlorella semenenkoi IPPAS C-1210 and Desmodesmus armatus ARC-06 were compared with the model strain Chlorella sorokiniana IPPAS C-1) cultivated at laboratory scale in flat-panel (5-L) photobioreactors. Biomass productivity, protein, starch and pigments contents, and CO2 utilization efficiencies were compared. The study shows a whole set of experiments and results that justify the objective of identifying optimal cultivation conditions for accumulation of different metabolites in selected strains with possible biotechnological potential.

The scope is both original and well organized. Figures (3) and tables (1) effectively illustrate and support the study. Supplementary materials (4 Figures) information is interesting, relevant and can help to understand the experimental set-up and the results obtained.

Methodologies and experimental set-up are not deeply described and should be improved and clarified according to the next comments.

Introduction: Page 2, line 57: …strain has been recently isolated and identified for biotechnological purposes.

Materials and methods

Page 3, line 91: …and under continuous 2700-3000 K LED…

Reviewers comment (RC): Was this strain ARC-06 cultivated under continuous light conditions?

Line 95: …C-1210 were grown in…

Paragraph 2.3. Flat-Panel Photobioreactors – RC: Please, could you describe how PBRs were inoculated with every strain? (i.e. starting biomass concentration according to description in Table 1)

Line 115: …70 mmol photons m-2 s-1

Paragraph 2.6 Growth characteristics - RC: In my opinion the description of the methodology for growth determination is not clear. It is necessary to write a detailed clear description on how these measurements were done. Maybe it is well described in the reference Life 2022, 12(9), 1309, but not in other references cited, neither in the present article.

Lines 151-152. RC: Could you please separate in the text the sentence for pH and optical density measurements?. Could you please describe how optical density was measured (wavelength, equipment, etc.)? One determnation is pH and a different one is optical density

Line 152: RC: Could you please briefly describe how CUE was determined?

Paragraph 2.7. Biochemical composition – RC: Please, it should be interesting to describe the method for protein and starch determination. These methodologies are not clear enough in the original reference. For example: how do you differentiate structural from storage polysacharides? How do you separate starch from other carbohydrates? Using the Dubois method (phenol-sulphuric) will give a measurement of total sugars, not just sugars from storage/starch.

Line 166: RC: Were the extracts centrifuged before measurements for pigments quantification? Please explain.

Line 168: Ref 24 and 25 are Ritchie, 2006 and Wellburn, 1994… delete the text

Line 181-182: The resulting concentrated biomass paste, with a humidity of 65-75%, was placed in Petri dishes and…

RC: No statistics have been applied to the results obtained. Results are basically descriptive and no comparison and significance between strains growth, CUE, biochemical composition and culture conditions can be clearly stablished and discussed if any statistics have been worked.

Results. Paragraph Lines 214-218 - RC: Simplify this comment. If no statistics have been applied you cannot discuss about “statistical differences”, just describe if values are similar, higher or lower through the culture period.

Regarding results on protein and carbohydrate composition, differences between ARC-06 and the other two strains C-1 and C-1210 seems to be clear. However, it is also clear that the culture medium and culture conditions (temperature and light) were also different, so it is very difficult to stablish a conclusion regarding the biochemical composition of these strains.

It is also the same for growth characteristics, as it is discussed in the Discussion section

Line 257: RC: No comments should be done regarding statistically significant differences. Just describe the differences observed.

Finally, the document and its structure meet the standards generally accepted by the journal. The article is scientifically sound and provides interesting insights into the field of microalgal biotechnological characterization. However, according to the comments regarding methodologies and results as presented, in its present form, this manuscript do not meet the standards for publication in the journal Phycology (MDPI) and should be considered after MAJOR REVISION.

Comments on the Quality of English Language

English grammar and scientific language should be revisited.

Author Response

Dear Reviewer, thank you for your time and valuable comments. We tried to answer on your concerns. Please find the detailed responses in the attachment and in revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

phycology-3756717 

Type of manuscript: Article

Title: Productivity and Carbon Utilization of Three Green Microalgae Strains with High Biotechnological Potential Cultivated in Flat-Panel Photobioreactors

The article is under consideration for publication in a special issue of the journal "Development of Algal Biotechnology". The study fully complies with the objectives of the special issue.

The authors investigated three species of green microalgae (Chlorella sorokiniana IPPAS C-1 as a model culture tested in the authors' previous study, Neochlorella semenenkoi IPPAS C-1210 and Desmodesmus armatus ARC-06) in laboratory 5-L flat-panel cultivation systems and characterized the biochemical composition and assessed the potential for introducing cultures into CO₂ sequestration technologies.

However, the work does not carry any novelty; the results of this article are not of the level of the Q1 journal. The role of 9 people in such simple study is unclear. Therefore, in order to consider it further, a thorough revision of the work is required.

The following questions arose during the review:

Methods: Section 2.1. Were the cultures maintained at room temperature, with or without antibacterial agents?

The algae were tested at different temperatures and on different media with different N loading. This will obviously result in different biomass yields.

Precultivation took 2 weeks, which is quite a long time. I raise the question of the correct growth conditions and the level of bacterial contamination of the cultures during such a long cultivation (the authors should provide data confirming the absence/insignificant presence of accompanying microbes).

Section 2.2. Please clarify the parameters of the inoculum preparation stage: medium volume, pH, and aeration values. After reading this, a big question arises regarding the inoculum and the cultivation itself. What are the optimal conditions for the strains?

The lack of pH control during cultivation casts doubts on obtaining quality data on growth (under growth conditions on nitrates and CO₂ bubbling).

At each stage, the authors used different temperatures, for example, 22, 27, 32, and 36°C for C. sorokiniana IPPAS C-1 and N. semenenkoi IP-94 PAS C-1210. This is a great stress for microalgae; the authors do not disclose the range used at all and do not consider the reorganization of cells.

Sections 2.6 and  2.7. A question arises regarding the calculation of the dry mass of the biomass. To estimate the dry mass, the samples must be incubated at a temperature of 105°C.

The authors should consider that the correlation between OD₇₅₀ and dry weight is non-linear due to a number of reasons (e.g., changes in pigmentation or cell size), so inferring biomass weight using the correlation is completely incorrect.

Plus, there is a doubt about the correct choice of the inoculum volume. The authors draw conclusions about the biomass yield while not considering what percentage of the seed cells it was.

The description of the study repeats the previous work of the authors (Optimization of CO₂ Supply for the Intensive Cultivation of Chlorella sorokiniana IPPAS C-1 in the Laboratory and Pilot-Scale Flat-Panel Photobioreactors). The authors must justify the inclusion of the Chlorella sorokiniana IPPAS C-1 in the study, since this is considered a repetition of published material.

Main recommendations:

Key words: when listing Chlorella; Neochlorella; Desmodesmus; green algae; microalgae – indicate the full name of the microalgae being studied, leave only “microalgae” in the list of key words, and also indicate the cultivation mode.

Introduction: I recommend expanding the introduction, focusing on the cultivation modes and their subtleties, parameters, and nutrient media. Authors should justify the choice of research objects.

Materials and methods: provide information confirming the taxonomic status of the studied cultures.

Section 2.6. Please indicate how growth and productivity parameters were calculated.

Section 2.9. I strongly recommend removing gaps (days when measurements were not taken) from the description and illustrations.

Results and discussion: I recommend transferring information on the parameters from Table 1 to the “Materials and methods” section. Considering that the cultures were grown under different parameters, do not present the data together for all three objects, but provide a summary of the parameters and data obtained for each culture.

4.3. Application Prospects: should either be used in the introduction or reorganized, as the authors speculate on the value of the objects based on data on metabolites that they themselves do not study in this work (in particular, lipids and polysaccharides).

Conclusions should be shortened.

 

Author Response

Dear Reviewer, thank you for your time and valuable comments. We tried to answer on your concerns. Please find the detailed responses in the attachment and in revised manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors evaluated Carbon Utilisation and Biomass Productivity of Three Green Microalgae Strains with High Biotechnological Potential in Flat‑Panel Photobioreactors. Below are my comments/concerns:

1. Cultivation was only conducted for 8 days; longer-term productivity, stability, and possible contamination issues under continuous cultivation were not addressed.
2. Environmental variables (e.g., varying light intensities, temperatures, or CO₂ concentrations) were not tested, limiting understanding of strain performance under fluctuating real-world conditions.
3. Consider rewriting the introduction to more clearly explain why these three strains were chosen and what their biotechnological applications are.
4. Some sentences could be simplified for clarity. For example: "The ARC-06 cells were characterized by the highest starch content (51%); this was twice higher compared to C-1 cells." could be rephrased to "ARC-06 cells had the highest starch content (51%), approximately double that of C-1 cells."
5. Expand on why high starch content in ARC-06 is industrially significant (e.g., potential for bioethanol or bioplastic production).

Author Response

Dear Reviewer, thank you for your time and valuable comments. We tried to answer on your concerns. Please find the detailed responses in the attachment and in revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This new version of the manuscript has been deeply revisited and my concerns, questions and comments about the original previous version have been clearified. In my opinion the manuscript has been improved, the information related to the experimental setup and the procedures have been completed and the results, discussion and conclusions sections show very interesting information to justify the results obtained through the study.

Still I have some minor comments to consider by authors:

Page 4; Line 296: …was measured at 750nm by the Genesys 10S UV-Vis spectrophotometer.

(RC) With this equipment, how can you get Abs values at 750 nm of 17.92, 10.9 or 7.55 (Table S1)? (linear range is up to 3.5 A at 260 nm)

Sample dilution is described (line 298) but linearity is lost. How did you obtain these values?

Change the term “probe” to “sample” through the text

Which were the k values used for each strain in equation (2, line 325). Why did you use two equations (1, line 307) and (2, line 325) to estimate Productivity? Just for comparison? This is confussing.

Page 9, line 602: The pigment contents after 8 days of cultivation is presented in Figure 3b (delete “of the cells of the studied strains”)

Please check line 2095-2096 for reference by Varela and Pereira. Ref 39? Reference number?

Author Response

Dear Reviewer,

We sincerely appreciate your positive evaluation of our work. We have carefully addressed all your comments and questions in the attached document for your consideration.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Productivity and Carbon Utilization of Three Green Microalgae Strains with High Biotechnological Potential Cultivated in Flat-Panel Photobioreactors” has been substantially improved by the authors. However, I still consider it pertinent to raise some comments and questions:

The author's response that the novelty of the work lies in testing new cultures is considered incomplete and I insist that this issue be taken seriously.

I have not received a clear answer from the authors regarding the duration of pre-growth adaptation (took 2 weeks). The authors should take into account that this directly affects the costs of cultivation.

I completely disagree with the authors regarding the parameters for determining dry weight. To determine dry weight, including plant material, a standard protocol is used which involves drying at 105°C until a constant weight is achieved. In particular, the Algal Biomass Organization protocol recommends filtering a specified volume of culture and drying at 105°C overnight (http://www.algaebiomass.org/wp-content/uploads/2010/06/ABO-Tech-Standards-MDL-V-3.0-FINAL-11-29-10.pdf). This process removes moisture, and the remaining weight is considered dry weight. The temperature used by the authors leaves a risk of considering residual moisture. Biomass dried at 80 °C can be used for evaluation of metabolite content, but not for an accurate assessment of dry weight.

Given the methods used to measure dry weight and algal growth (by correlation between OD₇₅₀ and dry weight), the results are unfortunately questionable.

The authors avoid and do not consider such an important parameter as pH, especially when testing scaling approaches for intensive cultivation. pH level has a significant impact on the metabolism of algal cells, and deviations from optimal values ​​can reduce culture productivity due to decreased enzyme activity, including the RuBisCO enzyme and other parameters that will directly affect the results of cultivation. And the authors' answer that the рН range of 6-10 is optimal for many algae is not only incompetent but also a rude answer to the reviewer. Unfortunately, the authors did not take this parameter into account and therefore did not provide an answer regarding the optimal pH value for the tested strains.

Provide data on the loss of key macronutrients to show if they are in sufficient supply for each strain.

Author Response

Dear Reviewer,
We sincerely appreciate your positive evaluation of our work. We have carefully addressed all your comments and questions in the attached document for your consideration.

Author Response File: Author Response.pdf