Batch Fermentation of Salt-Acclimatizing Microalga for Omega-3 Docosahexaenoic Acid Production Using Biodiesel-Derived Crude Glycerol Waste as a Low-Cost Substrate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors:

1. The abstract should clearly and unambiguously state the purpose of the work.

2. In the abstract it was written: „Optimum operating conditions were found to be 0.25 vvm and 50 gL^-1 glycerol corresponding to the maximum cell dry weight and DHA production.” What does “0.25 vvm” mean? What does “50 gL^-1” mean? Reading only the abstract, it's not entirely understandable. I believe that the content of the summary should be understandable. Please provide a description for the unit "vvm".

3. In lines 57 to 62 it is written: „Polyunsaturated fatty acids (PUFAs) are important long chain omega-3 polyunsaturated fatty acids, which has been shown to have beneficial effects on preventing human cognitive function, brain development and function, long chain omega-3 polyunsaturated fatty acids, which has been shown to have beneficial effects on preventing human cognitive function, brain development function, cardiovascular diseases, cancer, schizophrenia, and Alzheimer’s disease [19-25].” This is not the correct notation. Please reword.

4. Please explain the abbreviation BDWG. Once it is written “glycerol” and another time “BDWG”. Please elaborate on the abbreviation "BDWG". All abbreviations and designations should be self-explanatory. It is best if there is a list of abbreviations and designations in the work.

5. What does "w/v" mean? I understand what w/w or v/v is. I do not specialize in the research presented in this work. However, they are interesting to me and related to what I do. As a reader, I would like to understand the work. It will be good for work if all symbols and abbreviations are explained.

6. The description from lines 124 to 131 should be reworded.

7. In lines 57 to 62 it is written: „and (iv) after separation, other nutrients (mineral salts, nitrogen source, etc.), with additional water was added to adjust the nutrient level (including glycerol) to the desired levels.” All nutrients should be listed. What is meant by 'the desired levels'. What are the desired levels?

8. The way of describing the content of Figure 3 should be changed (description below the figure).

9. In lines 57 to 62 it is written: „This is supported by the fact that during the first two days, the fastest biomass increasing at 0.25 g L^–1 glycerol concentration was achieved compared to the other concentrations (data not shown); and (2) salt content. S. limacinum is a marine alga.”. The content is not understandable. Please reword.

10. Small tables that fit on one page should not be split and presented on two pages. The title line of table number 5 is difficult to read. This is the result of splitting the title line of this table into two pages.

11. The way the contents of figure 5 are labelled should be done differently. I would present the contents of figure 5 in two separate figures (5 and 6).

12. In chapter "4. Conclusions” has no specific conclusions in the research conducted. This chapter should be supplemented with the most important conclusions and observations regarding the work performed. Please write in the Conclusions what makes the research results presented in this work stand out from the research results presented in other works.

Author Response

For reviewer 1

Author Response File: Author Response.pdf

Reviewer 2 Report

 This research article deals with the production of docosahexaenoic acid from glycerol byproduct from biodiesel manufacture. The article is in accordance with the journal’s scope, however, there are serious points that must be improved:

Major issues

-        English must be revised. There are some inconsistencies and severe issues in some sentences.

-        The abstract must be revised to summarize the main ideas and the focus of this work. I think some parts might be removed since no specific information for this work is given.

-        Line 99. By “can be applied” you mean that it was applied in this work or that could be extrapolated to other works at larger scale? That idea must be specified in this sentence.

-        Line 135. What do you mean with “inoculated at a rate of 5% (v/v) and incubated at a rate of 5% (v/v)”?

-        Lines 141-142. Why a 30% medium corresponded to an aeration rate of 0.25 vvm. I think this sentence lack sense.

-        Materials and methods must be rewritten since the information is explained in a way that may provoke misunderstandings.

-        Figure 1. The abbreviation for hours is h, not hr. Same for the rest of the manuscript. The numbers of each point must be erased, they can already be seen from the axis.

-        Line 173. What do you mean with “corresponding to its morphology”.

-        Information from table 1 and figure 3 can be explained in the text rather than using table and figure, or at least, as supplementary material.

-        In general, the idea and objective of this paper is very interesting; however, the explanation is poorly performed. It is so hard to follow all the process, especially the materials and methods, and the results and discussion sections. The paper must be practically rewritten to carefully explain all the processes performed and the obtained results must be clear for everyone reading this paper. In my case, nothing was clear, at all. I will give no comments on the results section since I am expecting a thorough remodel for the following revision (R2).

-        Conclusions must be revised to highlight the novelty of this paper in comparison with others.

Minor issues

-        Line 41-42. Something is missing in that sentence. Maybe it could be modified to “for conversion of glycerol” or “to convert glycerol”.

-        Revise line 57-62. The sentence is repeated and lacks sense.

-        Line 62. Categories?

-        Line 67. “…has received”.

-        Line 68. “Recently,” or “In the last decades,”.

-        Line 94-95. The font seems different here. Same for lines 170-171.

-        Line 94. The abbreviation BDGW is not defined.

-        Line 134-135. Revise that last part of the sentence since it makes no sense.

-        Line 156. Either a starting bracket is missing of a final bracket should be erased.

-        Line 162. Flame or FAME?

-        Line 201. Erase the spaces.

Moderate editing of English language required.

Author Response

For reviewer 2

Author Response File: Author Response.pdf