Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

No items were found that need correcting or further explanation.  Article is very well written and all technical aspects are well supported and explained in the document.

Author Response

Dear Reviewer,

Thank you for your positive and constructive review. We are very pleased that you found the article well-written and technically sound.

Reviewer 2 Report

Comments and Suggestions for Authors

The article is well-written and meets all the requirements for a scientific paper. The research topic is both relevant and interesting, as it allows for the development of a method for culturing microalgae to produce biomass with maximum efficiency. This can be used in pharmacology, the food industry, and in the development of wastewater treatment plants.
The authors describe in great detail all the methods they use in their work for a multifactorial study of Chlorella sorokiniana culture. The results obtained and the conclusions drawn from them are beyond doubt. The work can be published with minimal revisions.
Nevertheless, I have several questions and comments regarding specific aspects of the article, which I have noted below.

Page 3: "The algae inoculum was grown aseptically in two or three vessels." So, two or three vessels? It's better to specify a specific number.

Page 3: "Glass aquarium with internal dimensions of 361 × 40 × 460 mm (L x W x H)." Would it be better to specify the larger dimensions first? This would make more sense, since 361 x 460 is the area through which the reactor is illuminated, and 40 is its width, i.e., the optical path length.

Page 4, "The optical density of the suspension was measured at 750 nm by the Genesys 10S UV-Vis spectrophotometer." Why was this wavelength chosen for optical density analysis? In the article [44] cited by the authors when describing the subtleties of the OD measurement technique, for example, Chlorella vulgarus was studied at a wavelength of 550 nm.

Page 4, "Using the correlation coefficient (k) between OD750 and biomass concentration," in the supplementary material, the authors provide references to articles describing how to obtain the correlation coefficient. Perhaps they should also be cited in the text of the article at this point?

Page 6: "Before measurement, the cultures were diluted to approximately 0.2 OD750 in distilled water." Dilution with distilled water can cause stress to microalgae, which has a high chance of being reflected in changes in their photosynthetic activity. At a minimum, such dilution should affect the pH of the medium and the concentration of dissolved salts. Can we say that dilution does not significantly contribute to the measured photosynthetic activity?

Figure 1b shows a significant decrease in the efficiency of carbon dioxide aeration, which, according to the authors, is associated with a decrease in cultural growth rates. If the carbon dioxide concentration during aeration is increased after day 3, can growth rates be restored?

Page 15: "This is evidenced by the total biomass obtainable on day 5: 31.82±2.2 g DW from our system versus 20.4 g DW from 1.8 L Labfors 5 PBR. Thus, our reactor design prioritizes total yield over peak volumetric productivity, resulting in a higher overall yield." A dubious statement. Perhaps it is more accurate to estimate the specific gravity of biomass rather than the absolute value. Moreover, could the limitations associated with a lack of light due to a long optical path be addressed by stirring the culture or using cold light, which has greater penetrating power in water than warm light?

Page 15: "The reported average maximum biomass yield for the 10-40% dilution range (1.634-1.662 g L⁻¹) exceeded that achieved in batch cultivation (1.564 g L⁻¹). The excess is insignificant. What does "average maximum" mean in this experiment? If an optimum was sought, what was it? Why were different dilution ratios chosen in your experiment, which essentially make it impossible to use the results of [31]? For the dilutions of 50, 75, and 82.5% used, an optimal number of days for harvesting was not determined. Such a study could significantly strengthen the argumentation for the final selection of cultivation parameters.

An economic assessment of the results obtained would be of interest, namely the profitability of cultivation in the proposed form, the ratio of the cost of the obtained product to the cost of the resources expanded, in the form of a nutrient medium and bioreactor energy to maintain optimal conditions.

Author Response

Dear reviewer, thank you for your time and valuable comments. We tried to answer on your concerns. Please find the detailed responses below.

Comment 1. Page 3: "The algae inoculum was grown aseptically in two or three vessels." So, two or three vessels? It's better to specify a specific number.

Response 1. Thank you for raising this point. To clarify, the study comprised five independent experimental series conducted over several months. In each series, biomass was pre-cultured in LSIC vessels, pooled into a single homogenous volume, and its density measured before equal inoculation into the main reactors. The number of pre-culture vessels (either two or three) varied by series, but as the biomass was always pooled and standardized, this variable does not affect the experimental outcome. We have therefore removed this specification from the manuscript to avoid ambiguity.

Corrected as: “The algae inoculum was grown aseptically in vessels of the laboratory system for intensive cultivation as described before [38]…”

Comment 2. Page 3: "Glass aquarium with internal dimensions of 361 × 40 × 460 mm (L x W x H)." Would it be better to specify the larger dimensions first? This would make more sense, since 361 x 460 is the area through which the reactor is illuminated, and 40 is its width, i.e., the optical path length.

Response 2. Corrected as suggested by the reviewer

Comment 3. Page 4, "The optical density of the suspension was measured at 750 nm by the Genesys 10S UV-Vis spectrophotometer." Why was this wavelength chosen for optical density analysis? In the article [44] cited by the authors when describing the subtleties of the OD measurement technique, for example, Chlorella vulgarus was studied at a wavelength of 550 nm.

Response 3. 750 nm is the standard wavelength for monitoring biomass and crop density in algology. Measuring at 750 nm minimizes the influence of cellular pigments (chlorophylls and carotenoids), which absorb light at other wavelengths. In this case, light scattering by cells is measured primarily, which correlates with their concentration and the dry weight of the biomass. (Solovchenko, A. (2023). Seeing good and bad: Optical sensing of microalgal culture condition. Algal Research, 71, 103071 https://doi.org/10.1016/j.algal.2023.103071)

Comment 4. Page 4, "Using the correlation coefficient (k) between OD750 and biomass concentration," in the supplementary material, the authors provide references to articles describing how to obtain the correlation coefficient. Perhaps they should also be cited in the text of the article at this point?

Response 4. Corrected as suggested by the reviewer. The corresponding references were added.

Comment 5. Page 6: "Before measurement, the cultures were diluted to approximately 0.2 OD750 in distilled water." Dilution with distilled water can cause stress to microalgae, which has a high chance of being reflected in changes in their photosynthetic activity. At a minimum, such dilution should affect the pH of the medium and the concentration of dissolved salts. Can we say that dilution does not significantly contribute to the measured photosynthetic activity?

Response 5. Thank you. We acknowledge that any sample manipulation can theoretically influence the measured parameter. In our specific case, the original culture had a near-neutral pH and low salinity (<4 g/L), minimizing the risk of significant osmotic shock during the brief measurement period. The primary purpose of dilution was to standardize the optical density (~0.7 OD750) for accurate O₂ evolution measurements and prevent signal saturation. Crucially, an identical dilution protocol was applied to all samples immediately prior to measurement, ensuring that any potential dilution effect was systematic and did not bias the comparative analysis of photosynthetic activity between experimental samples.

Comment 6. Figure 1b shows a significant decrease in the efficiency of carbon dioxide aeration, which, according to the authors, is associated with a decrease in cultural growth rates. If the carbon dioxide concentration during aeration is increased after day 3, can growth rates be restored?

Response 6. We are grateful for your detailed feedback and are pleased to address your question. We suggest that any potential increase in productivity under this scenario would be negligible for the following reasons related to our methodology and the physiology of the culture:

The key point lies in our definition of this parameter (Equation 4 in the text). We calculate it as the Carbon Use Efficiency (CUE) – the fraction of carbon from the introduced CO₂ that is fixed into biomass. This is a relative measure dependent on two variables: the rate of biomass accumulation and the total mass of introduced CO₂. Increasing the inlet CO₂ concentration raises the denominator in this equation, which, all else being equal, decreases the calculated CUE value, even if the absolute biomass gain remains unchanged.

In other hand a fundamental physiological limitation exists. By day 3 of cultivation in intensive cultivation the culture reaches a high cell density. Under these conditions, growth becomes limited not by carbon (CO₂) but by the available light energy (PAR). The photosynthetic apparatus of the cells is saturated, and additional substrate (CO₂) cannot be efficiently assimilated without an increase in light energy (according to Liebig's law of the minimum). Therefore, increasing the CO₂ concentration after day 3 would most likely not restore growth rates but would instead lead to its excess and venting from the reactor.

Comment 7. Page 15: "This is evidenced by the total biomass obtainable on day 5: 31.82±2.2 g DW from our system versus 20.4 g DW from 1.8 L Labfors 5 PBR. Thus, our reactor design prioritizes total yield over peak volumetric productivity, resulting in a higher overall yield." A dubious statement. Perhaps it is more accurate to estimate the specific gravity of biomass rather than the absolute value. Moreover, could the limitations associated with a lack of light due to a long optical path be addressed by stirring the culture or using cold light, which has greater penetrating power in water than warm light?

Response 7. We fully agree that for a fundamental comparison of physiological performance, specific volumetric productivity is the key parameter. As stated on page 15, we explicitly report and acknowledge the higher specific productivity of the thin-layer system (3.4 g DW L⁻¹ d⁻¹) compared to our setup (1.52 g DW L⁻¹ d⁻¹), attributing it correctly to the shorter light path. Our concluding statement about "total yield" aims to highlight a different, application-oriented perspective: for industrial biomass production, the total harvestable biomass per production unit and the scalability of the design are often more critical than peak volumetric density. A very short light path, while excellent for lab-scale density records, creates a significant scale-up challenge for working volume. Our data shows that our configuration, even with a lower volumetric productivity, delivers a higher absolute output per reactor run (31.82 g vs. 20.4 g), which is a relevant result for process development.

Regarding "cold light" (e.g., LEDs with a bluer spectrum): While certain spectral ranges can exhibit greater penetration in water, a significant shift towards a "colder" spectrum is unlikely to yield a major improvement in our system. This is because efficient photosynthesis and active culture growth require a balanced spectrum, including a substantial proportion of red light (around 600-700 nm), which is crucial for photosystem II and chlorophyll absorption. Therefore, optimizing the spectrum for penetration alone, at the expense of photosynthetic efficiency, would be counterproductive.

Regarding culture stirring/mixing: We fully agree with the reviewer that this is a critical and highly promising avenue for improvement. Enhanced mixing directly addresses the core limitation of a long optical path by actively transporting cells from dark, light-limited zones to the illuminated photic zone near the reactor walls. This can significantly increase the time-averaged light availability per cell, thereby boosting overall productivity. In fact, advanced mixing strategies—such as creating specialized liquid or gas flow patterns (e.g., using guided baffles, airlift loops, or swirling flows)—coupled with computational fluid dynamics (CFD) modeling to optimize these patterns, represent a forefront of reactor engineering. Implementing such solutions is a recognized and important direction for the future development of our system. Targeted hydrodynamic optimization through improved mixing is a powerful strategy we intend to explore further to mitigate light gradient limitations.

Comment 8. Page 15: "The reported average maximum biomass yield for the 10-40% dilution range (1.634-1.662 g L⁻¹) exceeded that achieved in batch cultivation (1.564 g L⁻¹). The excess is insignificant. What does "average maximum" mean in this experiment? If an optimum was sought, what was it? Why were different dilution ratios chosen in your experiment, which essentially make it impossible to use the results of [31]? For the dilutions of 50, 75, and 82.5% used, an optimal number of days for harvesting was not determined. Such a study could significantly strengthen the argumentation for the final selection of cultivation parameters.

Response 8. We thank the reviewer for the opportunity to clarify our methodology. Our statement directly references the specific results from the cited study [31, p. 7], which reports: “The average maximum biomass obtained at the end of each cycle of cultivation with a renewal rate of 10, 20, 30, and 40% were 1.634, 1.658, 1.662, and 1.627 g·L⁻¹, respectively, which were higher than the maximum biomass of 1.564 g·L⁻¹...”. Definition of "average maximum": in this context, the term refers to the mean value of the peak biomass concentrations recorded at each steady state for the four tested dilution rates (10%, 20%, 30%, and 40%).

The primary objective of this part of our work was to identify a practically viable semi-continuous regime for stable, long-term cultivation. We selected higher dilution rates (50%, 75%, 82.5%) to evaluate their feasibility from a bioprocess engineering perspective. Our goal was to move beyond optimizing solely for peak volumetric productivity and instead find a regime that balances yield with operational efficiency. A higher dilution fraction extends the time between harvests from hours to days, reducing the frequency of downstream processing could lower associated energy and operational costs, which is critical for scale-up.

For these higher dilution rates, the harvest trigger was based on the culture reaching the optimal biomass concentration window observed during the linear growth phase in batch mode. We acknowledge the reviewer's valid point that a systematic study to determine the optimal harvest day for each dilution rate was beyond the scope of this study. Such a detailed kinetic optimization is an excellent suggestion and constitutes a key objective for our further research as we prepare for implementation in a novel production facility.

Comment 9. An economic assessment of the results obtained would be of interest, namely the profitability of cultivation in the proposed form, the ratio of the cost of the obtained product to the cost of the resources expanded, in the form of a nutrient medium and bioreactor energy to maintain optimal conditions.

Response 9. We agree with the reviewer that a comprehensive economic assessment is a crucial next step for evaluating the industrial potential of the proposed cultivation regime. In response, we have performed a preliminary analysis of direct operational expenditures (OPEX) for one cultivation cycle in our experimental setup to provide a foundational data point and highlight the complexity of a full techno-economic analysis (TEA).

Our calculations for a single 17-day semi-continuous cycle (total working volume of 31.25 L, yielding 114.37±3.28 g DW) are as follows:

• Nutrient medium: ~8 RUB/L (retail price for small volumes), totaling 250 RUB per cycle.
• Electricity: Average cost of 6 RUB/kWh. Total consumption for lighting, mixing, aeration, temperature control, and downstream processing (centrifugation, drying) was ~252 kWh, costing 1,512 RUB.
• CO₂ supply: ~330 L consumed per cycle. At a cylinder cost of 24,100 RUB for ~12,000 L of CO₂, the gas cost is ~663 RUB.
• Process water: ~550 L for filling, cleaning, and cooling at 65.8 RUB/m³, costing ~36 RUB.

The total direct operational cost for the cycle is approximately 2,461 RUB. This translates to a production cost of about 80 RUB/L of culture volume or 21.5 RUB/g of dry biomass.

We acknowledge that this is a simplified OPEX snapshot. A complete TEA, as rightly suggested by the reviewer, would require a dedicated studies as capital expenditures (CAPEX), scale-up effects, product-dependent revenue and its market price with other: labor and maintenance costs.  

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the article were able to confidently and fully answer all the questions posed during the peer review process, and also made revisions to the article where necessary.
I believe this work has undoubted scientific value and should be published in the journal Phycology.

In carrying out their further work, I would recommend that the authors pay attention to the comment made in their responses and take it into account.

Comments on "Response 7."

Still, the main difference that allows you to achieve an absolute biomass advantage over your competitors arises not from the reactor's features, but from the larger volume of the culture medium (5 liters versus 1.8 liters).

"This is because efficient photosynthesis and active culture growth require a balanced spectrum, including a substantial proportion of red light (around 600-700 nm), which is crucial for photosystem II and chlorophyll absorption."
However, blue light with wavelengths of 430–470 nm, which has a significant contribution to the spectra of “cold” LEDs, is also effectively absorbed by chlorophylls (a, b, c) and photosynthetically active carotenoids (MacIntyre H.L., Lawrenz E., Richardson T.L. Taxonomic Discrimination of Phytoplankton by Spectral Fluorescence // Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications / ed. Suggett David J. et al. Dordrecht: Springer Netherlands, 2010. P. 129–169.)

Author Response

Dear Reviewer,

We are grateful for your thorough review.  We agree with your comments and will certainly incorporate these valuable insights into our works.