Mass Cultivation of Microalgae: I. Experiences with Vertical Column Airlift Photobioreactors, Diatoms and CO₂ Sequestration

Round 1

Reviewer 1 Report

This manuscript presents a long-term demonstration of outdoor production of marine diatom using different PBR configurations. It will be certainly of interest to those in the field of algal biotechnology. Below are some comments for the authors: 

1. Please provide data on pH levels during the operation of PBRs. How much of pH difference was observed with the use of flue gas? It may be possible that a lower pH level in a flue gas-treated reactor contributed to the culture maintenance against biocontaminants.
2. It was interesting to know that NOx present in the flue gas likely replaced some portion of nitrogenous nutrients. If possible, please provide more discussion in terms of N mass balance (e.g., how much of NOx present in the flue gas seemingly ended up in the harvested biomass). 
3. There isn't any data point in Figs 15 and 16. Please revise them. 
4. Please provide more discussion on Fig 14. What does PCA analysis suggest other than light isn't the only limiting production factor? 
5. The authors insisted that the ions from galvanic anodes mounted on lights may have interfered with cellular production. This claim requires supporting data. 
6. The authors claimed a high light utilization efficiency in their reactors. This seems to contradict their explanation on a likely decrease in Rubisco synthesis (Lines 828-830). Overall, the comment on the nitrogen balance above will need to be addressed before making any statement on Rubisco. 
7. The authors mentioned in conclusions that economic analysis was performed based on this project. How did economic outlook look like assuming the use of the produced diatom biomass as a fish (or animal) feed? 
8. Some sentences are missing references. Please also double check for typos and grammar errors.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The Manuscript “Mass cultivation of microalgae: I. Experiences with vertical column airlift photobioreactors, diatoms and CO2 sequestration” requires revision before accepted for publication. The specific comments are given below.

1. In Introduction also mention the limits on the use of microalgae

https://doi.org/10.1016/j.scitotenv.2020.142168

2. In the introduction, mention microalgae-based photobioreactors for CO2 capture

https://doi.org/10.1016/j.jcou.2021.101581

3. Use of microalgae for wastewater bioremediation has been tested in many different

types of effluents, such as municipal, industrial, food and livestock – state whether other researchers obtained satisfactory results. It is worth mentioning items such as:

https://doi.org/10.3390/pr8050517,

https://doi.org/10.1016/j.scitotenv.2019.135303,

https://doi.org/10.3390/en13092186

4. Please indicate the manufacturer, city, country when mentioning the equipment.
5. Statistical analysis is very important in experiments! Why did the authors completely abandon these analyzes?
6. Table 8 - Is italics necessary?
7. Extend your conclusions with the most important results (numerical values).
8. Little cited literature from 2017-2021. Refresh references.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript reveals an interesting approach of up-scaling the growth of diatoms, a promising microalgae to apply, for instance, as aquaculture feed. The use of factory fume as nutrients supply, namely to avoid the release of CO₂, is interesting and aligned with worldwide guidelines in order to find strategies to avoid the emission of GHGs. Despite the great workload and amount of results displayed, the authors should perform profound changes in the manuscript to meet publishing minimum standards. These are my major suggestions:

1 - A careful revision of the English level and sentences’ structure should be performed throughout the whole document in order to significantly improve the manuscript.

2 – A more organized structure and objective speech should be followed in order to better guide the reader throughout the work.

3 – In the Abstract section it is mentioned, as part of the results, some results related to microalgal protein digestibility and absorption or the detection of an antifouling agent. However, looking at the results, this is insufficiently supported. For example, there are no in vitro or in vivo studies that prove these statements. The fact of having a specific amino acids per se does not mean that the protein will succeed in digestibly or bioavailability tests.

4 - The content of the Introduction section should be reorganized. For instance, the goals of the work are a bit scattered along the Introduction. I suggest that the authors place a paragraph at the end of this section highlighting their main objectives and clearly mentioning what makes this work distinctive from others already existing in the literature.

5 - Discussion poorly explored. There is a lack of data/studies cited to support the results obtained, being the explanations sometimes vague. Moreover, there are some references to (still) unpublished works of the authors that make impossible to validate the information of the present manuscript.

6 - The Conclusions section is poor and highlights data/results that are not described in the present manuscript.

 

Additionally, these are my minor suggestions:

1 - A careful uniformization of the decimal numbers should be performed throughout the manuscript. It is common to find, for example, “0.7” and “0,05”. The correct form is “0.7”.

2 - The citation of references is not correct sometimes (e.g., (Tomas 1997) should be (Tomas, 1997), Tibbetts, et al. should be Tibbets et al.). Additionally, there are some references that were not included in the list of references (the abovementioned Tomas 1997, for instance). Please check and correct these situations throughout the manuscript.

3 - Attention must be paid to the units of certain parameters. The correct format is mg.ml^-1 and not mg ml_-1 as happens in some situations. Please do a complete revision of the units’ format.

Line 26 – Please correct to “The content of heavy metals…was well…”

Line 28 – Please correct to “…several promising findings.”

Line 38 – Since there is no other mention in the text of the terms CCS and CCU, they should not appear as abbreviations.

Line 50 – Is it “several” or “100”?

Lines 52 & 53 – Please correct to “…to be future’s large producers…”

Line 61 – Please correct to “We here have focused on…”

Lines 65-67 – Please rephrase the sentence

Line 71 – Please correct to “…is on wait, according to Collins et al. [26].”

Lines 72 & 73 – Please correct to “Despite that, they are…”

Line 86 – Please correct to “…process are troubles with…”

Line 105 – Please correct to “…also play a role…”

Lines 109 & 110 – Please correct to “Cermeno et al. (2005) [52]…”

Lines 113-117 – All the genus and species of microalgae/diatoms should be in italics.

Line 128 – Are referring to maximal biomass concentration, productivity, or both? It is not clear.

Line 137 – Please correct to “…simply be that algae concentration inside reactors are…”

Lines 138-139 – Please correct to “…will absorb and scatter more light than…”

Lines 140-141 – Please correct to “…modified by the fact that microalgae cells consist of…”

Lines 173-175 – This sentence is a bit confusing. Please rephrase it.

Lines 175-176 – Please correct to “When choosing the reactor type, large volume, low cost and area efficacy were the guiding principles.”

Line 180 – Please correct to “In the recent years,…”

Line 231 – Please correct to “…nutrients added were adjusted…”

Table 2 – pH is not a unit

Line 248 – Please correct to “…reactor were of the…”

Lines 261 & 262 – This information is repeated above.

Line 268-271 – There is something missing in both sentences.

Lines 271-272 – Could you please indicate the methodology used for cell size measurement? That information is not clear from Table 2.

Line 276 – Please correct to “Occasionally, in vitro…”

Lines 299 & 300 – Please correct to “From middle 2020,…”

Line 308 – Please correct to “…diffuse radiation might be obtained.”

Line 332 – Please correct to “In vitro Chla was…”

Line 353 – Could you please indicate how do you obtain the values “210” and “22.08” and what are they referring to? It is not clear for me based on what you describe.

Line 436 – Shouldn’t it be triple quadrupole mass-spectrometry?

Line 458 – Please correct to “…was freeze dried and extracted three…”

Lines 479 & 480 – Please correct to “…described in Osvik et al. [95].”

Lines 484 & 485 – Please correct to “…described by Osvik et al. [95]...”

Lines 490 & 491 – The expression “as described” is repeated in this sentence.

Lines 501 & 502 – Please correct to “Regarding the diabetes assay (PTP1B), both assay method and procedures are described in Ingebrigtsen et al. [98], while…”

Table 3 – This Table is a bit confusing. What is the meaning of the negative values here? Please change “vertikally” to “vertically” in the last column.

Figure 5 – Wouldn’t it be possible to present the standard deviation for each month to have an idea of the variation between different years?

Lines 530 & 531 – Please correct to “…varied from 3 to 100…”

Figure 9 – Please rephrase the subtitle.

Line 561 – Please correct to “…as doublings day^-1 are here shown…”

Figure 10 – Please correct to “…as biovolume pooled from…”

Lines 578 & 579 – Please correct to “…reached at maximum ca. 1.2 cm³ biovolume L^-1 during…”

Lines 582 & 583 – Please correct to “…reactor were in…resulted in an uptake of…”

Lines 607 & 608 – Please correct to “…factory smoke were observed…”

Lines 616-618 – Please correct to “All amino acids, except aspartic acid and arginine, presented higher values when cultivated with factory fume addition than with air (Table 8). The total DW specific amino acid content was therefore 34 and 30 % weight based when fume was and was not added, respectively.”

Table 8 – Please correct to “…(TAA) and total protein (mg g^-1 DW, n = 2) of biomass cultivated…”. All the other parameters than total protein (i.e., EAA and NEAA) are also quantified in terms of mg g^-1 DW? That is not clear here.

Table 10 – At the bottom of the Table there is repeated information regarding the concentration of As, Pb, etc.

Figure 15 – Please correct to “…reported in Osvik et al. [95] and Ingebrigtsen et al. [98]. Results…reported in Osvik et al. [95]. Color…”. What is the “fig. 2x for Flash/HPLC fractionations” you mention here? Please verify the graphs of Figure 15 because it is impossible to retrieve any kind of information from those.

Figure 16 – Please correct to “…reported in Osvik et al. [95], and HPLC fractioned extracts tested in A2058, FRAP, PTP1B and TNF_THPAIF/IM as in Ingebrigtsen et al. [98]. The figure…”. As happens for Figure 15, please verify the graphs of Figure 16 because it is impossible to retrieve any kind of information from those.

Line 662 – Please correct to “…part of future’s fish feed,…general, has to be…”

Lines 664-666 – Please rephrase the sentence since it seems incomplete.

Line 686 – Please correct to “…light were not limiting.”

Lines 695 & 696 – Please correct to “…as well as obtaining reproducible…”

Line 726 - Since there is no other mention in the text of the term IPR, it should not appear as abbreviation.

Lines 805 & 806 – Please correct to “…reactors also need frequent…”

Lines 831-840 – Could you please clarify the reasons for the discrepancy between your results (and those from references [142,143]) and those lipid content intervals indicated by Fields and Kociolek [144]?

Line 848 – Please correct to “…quality are important.”

Lines 850 & 851 – Please correct to “…resources is usually described by different metrics taking into account factors…”

Lines 852-854 – The sentence makes no sense as it is. Please rephrase it.

Line 862 – I believe that you are referring to Table 8 and not Table 6.

Lines 865 & 866 – Please correct to “…quality control were analyzed…”

Lines 875 & 876 – Please correct to “…possible to assess…Nagarajan et al. [150]…”

Lines 886 & 887 – The sentence makes no sense the way it it.

Lines 889 & 890 – There might be something missing in this sentence.

Line 916 – What is the meaning of “(ref)” here?

Lines 933 & 934 – Please correct to “…extracts make it more…”

Line 947 – Please correct to “…large cells that allow for…”

Comments for author File: Comments.docx

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors addressed most of the comments raised by the reviewers. The manuscript seems to be ready for publication after checking for any minor typos and grammar errors. One specific comments left is that the data points in Fig 16 and 17 are still not visible (at least the manuscript received by this reviewer). Please double check this in the revision process. 

Author Response

Thanks for help

Reviewer 2 Report

Thanks for replying to my comments. I recommend the manuscript for publication. All the best! 

Author Response

Thanks for help

Reviewer 3 Report

The authors have significantly improved the manuscript, elevating the overall quality of the reported work. However, some minor English mistakes are still detected throughout the document. Additionally, to make this manuscript ready for publishing, the following minor questions should be addressed, in my opinion:

 

Lines 321-332 – If the estimated supplied fume (400,619 L) was performed for 24 hours, why do you multiply again by 24 to obtain the amount of CO₂? Shouldn’t it be just 400,619 L x 0.05? If that is the case, all the following estimations of this paragraph are incorrect.

 

Figures 16 and 17 – It is still impossible to retrieve any information from these Figures. Apparently, there is something missing (dots/colour?).

Author Response

Response to Reviewer 3 Comments Round 2

 

Point 1:  Lines 321-332 – If the estimated supplied fume (400,619 L) was performed for 24 hours, why do you multiply again by 24 to obtain the amount of CO₂? Shouldn’t it be just 400,619 L x 0.05? If that is the case, all the following estimations of this paragraph are incorrect.

 

Response 1: Oops, unforgivable, due to script used not tuned with text when revising. Led us to re-check all computations. Problem with present data set is that some computations of e.g. growth rates and production sometimes are over 2-3 days, i.e. production was continous but logging of data was during shorter periods. Though conclusive data is sum or means of this etc. What we have now more consequently implemented is that we have used 75 percentile method, i.e. only used data from percentile 1-2-3 and thereby considered “peaks” as outliers. This makes obtainable data more “trustworthy”. We have made this clear in the text. What concerns we have checked data sets, and in addition re-done an experiment done earlier where we also measured CO₂ pressure in culture and 1 m above surface of culture but well inside tank! New text here is then:

 

“Estimation of potential maximum amount produced of algae biomass from CO₂ in fume was calculated from several cultivation sessions from measured gas flow in pipes, CO₂ content in the fume (Table 2) and a algae DW : CO₂ conversion factor of 2, i.e. (time * flow * pipe area * CO₂fraction in fume). Example is: CO₂ uptake during 24 hours (86400s  * 105cm s^-1 * 44.16cm² * 0.05 = 20030L). 1L of CO₂ at atmospheric pressure has a weight of 1.84 g, then the added CO₂ can maximum be converted to 36.5kg biomass. Harvested biomass (DW) was 12 kg. From this the logged uptake efficiency was 33%. Applying measured CO₂ pressure in the culture and 2 m above culture surface (but well below rim of reactor) in similar situations yielded 709 ppmv in culture and 494 ppmv above, i.e. d ppmv was 215 ppmv. This method, since the measured fluxes were stabilized over time, yielded a CO₂ uptake efficiency of 30.03%.”

 

 

Calculation of light utilization has also been made more understandable (and conservative): “To estimate photosynthetic efficiency, it is necessary to include the energy content of the microalgae. Platt and Irwin [91] reported caloric contents of 2.151 to 3.529 calories mg^-1 DW in microalgae (diatoms) in a field spring bloom situation. Tibbetts et al. [92] reported 19 -27 MJ Kg^-1 dry biomass, i.e. 4.5 – 6.45 calories mg^-1 with the highest values in blue-green and green microalgae and the lowest in a diatom. We applied 2.1 calories mg^-1 (8.7 J) as a conservative estimate of the energy in our mass cultivated diatoms, this taking the measured protein and lipid content in consideration. Example computation with 1/3 of the large reactor full is: A mean production value of 0.28 g L^-1 day^-1 DW (see Results chapter) will therefore amount to ca. 280*8.7^*100 000/86400=2.85kW (1W=J s^-1) “produced” in 24 hours. During production peaks total irradiation in the reactor was 50 – 55 000 mmol m^-2 s^-1, i.e. equivalent to 14.39 kW using the conversion from Eilertsen and Holm-Hansen [93]. Light utilization was then 2.85/14.39%=19.8% relative to total light energy delivered to the reactor. The final efficiency was calculated with different volumes in the reactor (see Table 6).”

 

Also some other “improvements” are implemented in text.

Point 2: Figures 16 and 17 – It is still impossible to retrieve any information from these Figures. Apparently, there is something missing (dots/colour?).

 

Response 2: Oops, have imported as .pdf, seems to work!