Enhancement of Metabolite Production in High-Altitude Microalgal Strains by Optimized C/N/P Ratio

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1

After reading the introduction (line 58), it is expected that the authors have grown the algae in conditions equivalent to natural conditions. But I find that 100 µmol photons m-2 s-1 (line 77) or 100 µmol photons m-2 s-1 (line 94) is rather a low light intensity. Could the authors explain their choice?

Revised and changed, it was a mistake. The actual intensity for most metabolites was 100 µmol photons m^-2 s^-1, but for carotenoids it was 250 µmol photons m^-2 s^-1

Materials and methods

2

Moreover, what is the temperature? What is the pH?

Added  

76-78

95-96

97-98

3

Lines 80-82 : Why have carbohydrates and proteins analyzed in Chlorella, and lipids and carotenoids analyzed in Scenedesmus?

As stated in lines 72-73, the strains were chosen from a larger set of algae which belong to an unsubmitted paper

72-73

4

Why not the 4 biochemical compounds in both algae?

Each strain was chosen due to its ability to produce “more” of one specific metabolite. Sadly, DoE will only allow to enhance one metabolite since the C/N/P ratio will reduce the concentration of others metabolites.

5

Table 1, line 90 :

- NaNO3 and K2HPO4 + KH2PO4 should be expressed everywhere in g/L but not in ml/L.

- What are the proportions of K2HPO4 and KH2PO4 ?

Table modified with mL/L plus g/L  

Table 1

6.

Table 2 : What does (C) mean?

“C” means central point. Added also in table 2

Table 2

7.

Line 101 : the centrifuge force should be expressed in g but not in rpm.

Changed to “g”

105-106

8.

I am not familiar with pareto charts, and maybe other readers of this article will be too. So authors should explain briefly how to understand this kind of chart and why they have chosen it.  Fig 1a

- What do Sc and MS Residual mean?

- For Y axis, explain : (ml/L)(Q) and similar expressions; 1Lby2L and similar expressions; why it is written (3), (1) or (2) in front of the name of certain chemical compounds?

- Why are there negative values beside some bars?

- same remarks for similar figures through the manuscript Fig 1b, 2d, 2f, 3d : Why are there negative values? Table 3 : Add cells to make the table more readable.

Explanation added

111-118

References 114-118 are not appropriate. Please, replace them by references dealing with the optimization of culture media and extraction of different metabolites from microalgae.

Please, include :

Ho et al. Bioresource Technology Volume 135, May 2013, Pages 157-165

Lv et al. Bioresource Technology Volume 101, Issue 17, September 2010, Pages 6797-6804

Mandik et al. Bioresource Technology Volume 182, April 2015, Pages 89-97

Sánchez Zurano et al. Scientific Reports (2021) 11:21651

Tahiri et al. Annls Limnol. 36 (1) 2000 : 3-12

Ghosh et al. Environmental Science and Pollution Research, https://doi.org/10.1007/s11356-019-07115-5

and perhaps

VILLAR-ARGAIZ et al. Freshwater Biology (2001) 46, 1017±1034

Gunkel & Casallas, Limnologica 32, 33-43 (2002)

New paragraph added in the introduction and added

New references added 89-93

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1

The introduction denotes a rather long list of strains which can be used for cultivation and some comments that register why they are interesting for culture. The same is not underlined for the two microalgae under observation. Why are these strains important? In what manner are they more interesting to be cultivated? Do they carry any advantages in relation to commercial strains? Do they require specialized temperature/pressure control?

Introduction adjusted

introduction

2

There is an overabundance of references. Please limit them.

The other 2 reviewers requested the addition of more citations; therefore, we can’t comply with this request.

3

Why was not autotrophic production considered?

Initially, the use of glucose was considered, but it was finally decided to use sodium acetate as the only source of organic carbon, therefor the illumination was maintained.

4

Is there a reason why Scenedesmus wasn’t considered for carbohydrate accumulation? Scenedesmus obliquus, for example, is well known for being an excellent starch producer.

Each strain was chosen due to its ability to produce “more” of one specific metabolite. Sadly DoE will only allow to enhance one metabolite since the C/N/P ratio will reduce the concentration of others metabolites.

5

From line 189-216, it is effectively data that should be in the introduction and does not actively discuss the results presented in the previous section.

Information moved

introduction

6

Line 240-242, burst the final content? This sentence is not clear.

Phrase changed

discussion

7

A point of discussion to be addressed should be what the authors suggest would be the best option for metabolite production by the studied strains. Additionally, what are the yields attained? How do they compare to other strains? It would be interesting to know these in order to ascertain whether the cultivation of these microalgae would be better than other more commercially available strains.

Discussion adjusted

Discussion

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section

1

Section "Introduction" looks superficial, it is necessary to add information about the data of scientific works devoted to the study of the role of various factors, including the concentration of macronutrients on the metabolism of green microalgae. The authors did not touch upon the features of cultivation of the studied microalga to obtain target products. What conditions and nutrient requirements should be provided for green microalgae for their effective productivity? I advise you to add a few works in this area:

https://doi.org/10.3390/biology7020025;

doi.org/10.3390/biology9070169;

10.1016/j.btre.2017.11.009;

DOI: 10.1038/s41598-018-24979-8...and others

Introduction adjusted with new references

introduction

2

In the section "Materials and Methods" there is no information on the method of identification of the studied strains. Please add.

Method added,

Materials and methods

3

Please give the concentration of the all compounds as mg/L (no as mL/L) .

Units format homogenized

Tables in Materials and methods, and results

4

The level of illumination was 100-110 μmol m-2 s-1, but this level is low, which can be attributed to the low yield of the studied pigments.

Revised and changed, it was a mistake. The actual intensity for most metabolites was 100 µmol photons m^-2 s^-1, but for carotenoids it was 250 µmol photons m^-2 s^-1

Materials and methods

5

 Why the authors decided to study the optimization of the accumulation of carbohydrates and lipids in Chlorella cells, and of proteins and carotenoids in Scenedesmus cells? For example, many representatives of the genus Chlorella actively accumulate proteins, while representatives of the genus Scenedesmus accumulate lipids.

As stated in lines 72-73, the strains were chosen from a larger set of algae which belong to an unsubmitted paper

Materials and methods

6

L-87: Did the calculation of the final carbon concentration take into account the carbon content of the supplied carbon dioxide (1%)?

In the expriments belonging to DoE, the strains were grown without extra CO₂. Line upgraded

Materials and methods

7.

Please change "Bold Basal Medium" to "Bold's Basal Medium", and capitalize (e.g. line 179).

Name changed

document

8.

In the section "Materials and Methods" only references to the definition of metabolites are given. I recommend giving a short description.

Methods added

Materials and methods

9.

 The work lacks data on the growth characteristics of each strain under the tested conditions (in particular to characterize the growth of cultures under optimal scenarios).  Please provide specific growth rate and biomass productivity values.

Comparative table added in discussion

discussion

10.

Formatting advice: do not break the figures from each other (a-f) and from the name of the figure and modify table 3 (column 2 (metabolites) seems redundant, these values are mentioned in column 3).

As stated by other MDPI editors, all the figures must be separated. Table 3 upgraded

11.

Why do the authors study the effect of C/N/P only on carotenoids, are there any data on the effect on chlorophylls a and b?

The authors didn’t considered chlorophylls as a valuable metabolite, since most literature focuses on carotenoids in algae

12.

Figure 5: no standard deviations for controls.

Figure upgraded

results

13.

There are unnecessary capital letters in the text, for example lines 192, 201, 239, 256.....

Capital letters revised

Discussion

14.

In the "Discussion" section, the authors compare the results of their work with the works, the object of which are representatives of cyanobacteria, I would advise not to include this part or minimize it, these are different groups

Discussion upgraded

Discussion

15.

What is the number of replicates?

Three replicates per experiment in both production and extraction

Materials and methods

16.

Please add pH changes during cultivation of algae.

pH was only adjusted at the beginning, no pH control was done during the experiments

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1

Concentrations expressed in mL/L remain; please erase them or replace them by g/L everywhere (highlighted in orange or erased).

Revised and changed

document

2

Fig 1a : - What does “MS Residual” mean?

MS residual used in ANOVA In mean squares are used to determine whether factors (treatments) are significant.

The treatment mean square is obtained by dividing the treatment sum of squares by the degrees of freedom. The treatment mean square represents the variation between the sample means.

The mean square of the error (MSE) is obtained by dividing the sum of squares of the residual error by the degrees of freedom. The MSE represents the variation within the samples.

For example, you do an experiment to test the effectiveness of three laundry detergents. You collect 20 observations for each detergent. The variation in means between Detergent 1, Detergent 2, and Detergent 3 is represented by the treatment mean square. The variation within the samples is represented by the mean square of the error.

The MS residual was removed from each figure

3

For Y axis, explain : (Q) and (L) on Y axes; 1Lby2L and similar expressions; why it is written (3), (1) or (2) in front of the name of certain chemical compounds?

- same remarks for similar figures through the manuscript b.     

Explanation for the tags used by the software for Lineal (L) and Quadratic (Q) models, plus the number for each factor (1-3) was added in the first paragraph of results section

Line 160-171

4.

Table 2 : what does “central point” mean?   Some typos are highlighted in orange and corrected. Please find the detail in the attachment.

Center points are a statistical tool to estimate the pure error of the design and test for curvature.  Which in turn helps in the optimization process. If a design presents curvature the  model can be further optimized using only the most critical variables

1106-107

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section

1

Give the concentration of all compounds as mg/L or mM everywhere and update the figures and tables accordingly.

Revised and changed

document

2

Provide specific growth rate and biomass productivity values for each condition.

Results added for the optimal results,

results

3

In these experiments, the pH values had to be controlled necessarily, since they clearly did not stay at the level of the initial 7.0 and definitely affected both the growth characteristics and productivity.

Methods added

122-124

4

How efficiently were nitrates, phosphates and acetate utilized? Please provide data on the removal efficiency.

Method added and Figure added

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259-264