Investigation of Crypthecodinium cohnii High-Cell-Density Fed-Batch Cultivations
, Arturs Suleiko 1,2, Anastasija Suleiko 1, Elina Didrihsone 1, Mara Grube 3, Karlis Shvirksts 3 and Juris Vanags 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Based on the quality of this study. I do not think it is acceptable in the current status and thus a major revision is needed before it can be published in Fermentation. Please see the specific comments below.
I suggest revising the title to “Investigation of crypthecodinium cohnii batch cultivation with high cell density”.
Abstract
Basically speaking, the abstract fails in delivering the key results of this study.
Line 12 Change to “thus replace conventional routes of fish oil production that are associated with the destruction of marine resources…”.
Line 14 Change to “The major drawbacks of using C. cohnii for industrial scale production are associated with low DHA productivity”
Line 21 What is oxygen mass transfer?
Line 21 The biomass yields from individual components were determined from the experiment data. What does this mean? I think it can be removed.
Line 26 Please specify these conditions.
Line 29 You stated the DHA production using crypthecodinium cohnii in the beginning of introduction. But the results of DHA are missed in the abstract.
Lines 35-42 Here needs references.
Line 47-50 Here needs references.
Line 94 Higher CO2 concentrations can simulate the production of DHA. Please see https://doi.org/10.1007/s10811-021-02619-5.
Line 96 phosphorus and silicon also affect DHA synthesis in microalgae. Please see http://dx.doi.org/10.1016/j.foodchem.2015.08.073, https://doi.org/10.1016/j.biortech.2019.121717.
Line 222 For the results, why don’t I see the results of DHA since it is mentioned in abstract. In addition, a thorough discussion of your results is needed. Please compare your results with previous ones. I am also curious why using heterotrophic microalgae to produce DHA. Does it has a higher productivity compared to autotrophic microalgae? The culture cost of heterotrophic microalgae should be higher.
Line 248 Please place the units of conductivity, time and permittivity in brackets. Please explain what do the lines mean, particularly the dotted lines.
Line 294 The same comments as figure 2. Additionally, the coordinate axis for oxygen seems missed. I cannot understand which line represents glucose uptake curve.
Line 331 The same comments as figure 2 in terms of units.
Line 337 The explanation of inserted charts should be stated in the figure legend.
Line 395 Please also revise the units.
Line 409 The explanation of inserted charts should be stated in the figure legend.
Comments on the Quality of English Language
Language can be improved.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Review comments on “INVESTIGATION OF CRYPTHECODINIUM COHNII HIGH-CELL-DENSITY FED-BATCH CULTIVATIONS”
General comments:
Lines19-20: “The goal of the present research was to develop techniques for increasing the efficiency of DHA production via C. cohnii cultivation.” This sentence suggest increase yield of DHA is the main goal however, FTIR analysis does not conclusively suggest the increase of DHA due to applied growth conditions, which could even be EPA or other fatty acids of the total lipid composition. Justify. This needs extraction of fatty acids from the biomass and analyse the DHA content by one of the appropriate methods like HPLC, GC or LC-MS.
Specific comments:
Lines14-16: “Although, the major drawbacks of applications, which use C. cohnii for industrial scale production, are associated with low DHA productivity.” This sentence is in-complete. Re-write the same.
Lines20-21: “Multiple experiments were carried out to test and fine-tune the cultivation medium composition and oxygen mass transfer conditions.” Instead of using vague sentence with “Multiple experiments were carried out”, provide what was done.
Lines21-23: “The biomass yields from individual components were determined from the experiment data. The C. cohnii cell susceptibility to mechanical damage was experimentally evaluated.” Re-write above sentences providing data/results and not just general statement, which can anyways be seen reading the paper.
Lines25-27: “The obtained cultivation medium and feeding solution composition, as well as the aeration conditions showed themselves efficient in terms of increasing the biomass productivity and the omega-3 fatty acid content in C. cohnii.” Update the above sentences with real findings such as, mention the ‘cultivation medium’; what is that ‘solution composition’; what is that ‘aeration conditions’.
Line29: What PUFAs data you mean here based on FTIR analysis?
Line 58: Error in ref formatting “Patel et al. 2020” also check line 205: “Dubencovs et al. 2021”
Line123: “2.2. Bioreactor cultivations” What high-density culture amount used? “DCW = 10 g L-1” Is it final starting concentration in 4L medium? What would be low-density culture that any study would have used earlier? Can the data be comparable from low density culture, which is essential for the discussion to realise the effect of high-density culture in DHA production.
Line209: “2.6. Fourier transform infrared spectrometry” Add appropriate references for FTIR analysis of fatty acids in biomass.
Line214: Clarify what do you mean by “Omega FA”? Can FTIR analysis be FA specific?
Check for errors in Figure labels such as “Gllucose”
Line437: “From the FTIR data the presence of unsaturated fatty acids was confirmed.” Where is this result mentioned? Atleast mention here “What are the unsaturated fatty acids was confirmed”?
Line438: Be specific what “PUFA” you mean here?
Comments on the Quality of English Language
Minor typographical errors to be fixed
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
Reviewer report for the article
INVESTIGATION OF CRYPTHECODINIUM COHNII HIGH CELL-DENSITY FED-BATCH CULTIVATIONS
General comments:
Abstract needs more actual data obtained in the study. For example, you mentioned cultivation medium composition and optimal oxygen mass transfer conditions. Please provide values for mass transfer coefficient that was optimal for biomass and DHA productivity.
I suggest that you place the microalgae part (lines 63-75) at the beginning of the Introduction.
Line 67: “it (lipid content) can reach even higher levels (up to 85%) [7]” This is very high number and reference 7 is incomplete so it cannot be confirmed. Please remove or rearrange.
Line 71 “in previous studies” than you cite only 1 study. Please reconsider adding more sources.
Line 85 “Furthermore, the presence of more than 50 – 60% of fatty acids with 16 and 18 carbon atoms (C16–C18) in C. cohnii total fatty acid content makes this microalga a potential source for biodiesel production [5].” This is typical for many algal species as well or you claim it is unique feature of C. cohnii? Please clarify in the text.
Line 108: put alga name in italics please, also check it throughout the text…
Line 124: Please specify material bioreactor was made of.
Line 132: Did you use any antifoam concentrated or diluted? If yes, please specify in the text the concentrations applied per L of culture broth.
Line 204: “The biomass dry cell weight (DCW) in relation to the absorbance at a wavelength of 204 470 nm was determined gravimetrically as described by Dubencovs et al. 2021.” Citation has no number. Also please add short protocol for DW determination by stating - Briefly, …… (few sentences about Dubencovs method)
Line 212 “frequency 212 range of 4000 - 600 cm-1” It is typical to call them wavenumbers in FTIR technique. Please do so…
Line 220 “rubber band method” Is what? Please explain
Lines 237 and 243: Please use formula menu in your text editor to describe equations: 
From the values of the Figure 2 it seems you could use 250 rpm as well with the same effect. Please explain this?
Line 283 Figure lacks number
Line 289 “due to the depletion of sea salt components,” The first cultivation was feed batch or you also harvested some biomass? Where did sea salt go if it was batch? Please explain
Equation 5. Explain all used parameters, please. What is ms, for example?
Suggestion - please use h to describe time. Do not use both h and hours.
Lines 326-330: Please add elaborate discussion to these facts. What happened to the culture?
Line 355: Please remove “themselves”. Parameters are not live beings.
Line 397-399: This sentence belongs to introduction unless you immediately draw some conclusions.
Line 404-405: Numbers lack units. What is the parameter you talk about?
Figure 8. Although FTIR is a very good technique to study algal cell composition it is not quantitative. To use FTIR for some kind of quantification you would need to correlate spectra to DHA content and make some linear model.
Why did not You used any GC to determine DHA content and overall FA profile?
In the abstract you claimed: ”The goal of the present research was to develop techniques for increasing the efficiency of DHA 19 production via C. cohnii cultivation”
Nevertheless, actual DHA content was not measured only assessed by FTIR.
Can you perform GC analysis to determine FA profile and actual DHA content of some lipid extracts and add to the study to confirm the results?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I can see the authors revised the manuscript carefully based on my comments. I do not have further suggestions.
Reviewer 2 Report
Comments and Suggestions for Authors
Thanks for your revision but it could have been better you would have elaborated what are the Multiple experiments that were carried out
Comments on the Quality of English Language
Read the manuscript carefully for so many typographical errors specially in the new sentences during the revision.
Reviewer 3 Report
Comments and Suggestions for Authors
No further comments
