Variables Governing Photosynthesis and Growth in Microalgae Mass Cultures
, Karolína Ranglová 1, Gergely Ernö Lakatos 1, Ana Margarita Silva Benavides 3 and Giuseppe Torzillo 4Round 1
Reviewer 1 Report
The manuscript "Variables Governing Photosynthesis and Growth in Microal-gae Mass Cultures" by Jiří Masojídek et al. makes an overview of the recent progress in microalgal cultivation and growth monitoring with particular attention about the major constrains regarding photosynthethic productivity. The authors took efforts to submit a comprehensive review dealing with the different aspects of photosynthetic of microalgae mass cultures. In general, I found this review very informative in particular the section about photosynthesis monitoring is very useful.
I have only some advices for the authors about the section "Changing Antenna Size". Recently have been made many efforts in reducing PSII-antenna size in microalgae and at the same time by maintaing photoprotection by increasing ROS resistence in high irradiance. Maybe this section could be expanded.
Altought, recently it has been demonstrated that NPQ, one of the most studied photoprotective mechanism, is not involved directly in microalgae productivity, I advice to take in consideration to add a small section about NPQ in algae.
Author Response
Response to Reviewer 1 comments
Point 1: I have only some advices for the authors about the section "Changing Antenna Size". Recently have been made many efforts in reducing PSII-antenna size in microalgae and at the same time by maintaining photoprotection by increasing ROS resistance in high irradiance. Maybe this section could be expanded.
Response 1: In the literature we have found that truncated antenna may not be effective against reactive oxygen species (ROS) due to lesser ability to dissipate excess excitation energy via non-photochemical quenching (NPQ) process. Vecchi et al (2020) proposed that in the situation when the production of ATP and NADPH by the light reactions exceed their consumption rate by the Calvin-Benson cycle, and the overexcitation of PSII results in the release of reactive oxygen species (ROS), antenna system remodelling can be induced by slowing down the transcription of LhcbM genes (encoding the light-harvesting chlorophyll-a/b proteins of photosystem II).
The subchapter Changing Antenna Size was revised and extended (p. 8-9, lines 329-340) to discuss reduced PSII antenna vs. Photoprotection.
“Microalgae strains with reduced antenna size may suffer lesser photoinhibition at high light intensity, particularly when cell density is low, a situation that frequently occurs at the start of the cultures outdoors. However, as the culture become denser, and thus the available light is reduced, the productivity of the reduced antenna size strain is overhauled by WTs. Similar results were also obtained outdoor in thin-layer cascades comparing two Chlorella strains with different antenna size. Thus, a strain with the reduced antenna size is not sufficient to guarantee higher biomass productivity in mass outdoor culture.
Such mutants may also result in impaired photoprotection induced by antenna alterations and/or other undesirable side effects of genetic modifications such as increased respiration that reduces the benefit of the reduced light absorption in the truncated antenna cell. The protection of microalgae strain with truncated antenna again reactive oxygen species (ROS) may not be as effective due to lesser ability to dissipate excess excitation energy via non-photochemical quenching (NPQ) process.
Therefore, in these studies, an important variable that needs to be considered, other than the reduced PSII antenna size, is also the ratio of Pmax/Respiration which should not be significantly affected. Recently, a strain of Chlorella vulgaris g120 characterised by high respiration rate and low Chl content per cell was shown to perform much better than Chlamydomonas reinhardtii commonly used for photobiological hydrogen production.”
Point 2: Recently, it has been demonstrated that NPQ, one of the most studied photoprotective mechanism, is not involved directly in microalgae productivity, I advise to take in consideration to add a small section about NPQ in algae.
Response 2: We agree with the reviewer. NPQ (indicating dissipation of absorbed energy, mostly as heat) is considered as a protection mechanism of PSII reaction centres against damage by excess irradiance which is dissipated as heat. Photochemically fixed energy is used for biomass production. The role and components of NPQ were described in more detail in the subchapter 4.3 Chlorophyll Fluorescence Monitoring (p. 24, lines 1099-1125).
“The non‑photochemical quenching NPQ calculated according to the so-called Stern-Volmer formalism [= (FM - FM’)/FM’] is inversely related to the actual photochemical yield (YII or DF'/FM'). An increased NPQ (indicating dissipation of absorbed energy, mostly as heat) is considered as a protection mechanism of PSII reaction centres against damage by excess irradiance [197]. Semi-empirically, NPQ has been solved into two components – Y(NPQ) and Y(NO), which quantify regulated (DpH- and xanthophyll-regulated quenching in the light-harvesting antennae) and non-regulated (due to energy trapped in closed PSII reaction centres) thermal dissipation processes, respectively [147]. If summarised YII + Y(NPQ) + Y(NO) should equal 1.
NPQ arises from a number of processes in the thylakoid membranes, and several major components of NPQ can be identified based on the kinetics curves of the relaxation of PSII fluorescence [198]. The fastest component of NPQ, immediately triggered upon exposure to light, is the energy-dependent quenching (qE). State transitions represent changes in the relative antenna sizes of photosystems [47], however although this fluorescence decline (called qT) has been included in NPQ. An additional quenching component, that rises and relaxes at a longer time scale called qZ is found in some eukaryotic microalgae and plants [199,200]. The response is dependent on a low lumenal pH and requires Chl-xanthophyll-binding proteins. Under high-light conditions, lumen acidification triggers the so-called xanthophyll cycle, which involves the xanthophylls violaxanthin and zeaxanthin, and consists of a light-dependent, rapid and reversible de-epoxidation of violaxanthin to zeaxanthin [199,201]. The NPQ mechanism is highly relevant for the maintenance of the photosynthetic efficiency which contributes to acclimation to the different light environments. The relative contribution of each of the NPQ components changes between organisms and irradiances: qE activates based on sudden increases in light intensity, while ST responds to changes in the light spectrum under low light conditions [25]..”
Reviewer 2 Report
Please see attachment for details.
Comments for author File: 
Comments.docx
Author Response
Response to comment of Reviewer 2
There is frequent and unnecessary repetition of ideas (while thoroughness is an appreciated feature of this
manuscript the writing could be tightened up.
The texts were restructured to remove repetitions of facts and made the MS more concise.
Point 1: ideas in sentence on line 244 - 245 has already been mentioned in previous paragraphs
Response 1: The last paragraph of subchapter 3 (p. 6, lines 240-252) was restructured to remove repetition of the text of Figure 1 (p. 2).
Point 2: ideas in sentence on lines 250 - 251 has already been mentioned previously
Response 2: as in Point 1 – two paragraphs were merged.
Point 3: ideas in sentence on lines 265 - 268 has already been mentioned previously
Response 3: the text repetition was removed.
Point 4: ideas in sentence on lines 282 - 283 has already been mentioned previously
Response 4: this text is quite important to emphasize; we did not find it as being repeated.
Point 5: ideas in sentence on lines 395 - 400 has already been mentioned previously
Response 5: A part of the text on p. 9 (last paragraph) was removed to avoid repetition.
Point 6: ideas in sentence on lines 592 - 593 has already been mentioned previously
Response 6: this text is rather important to remain there and we did not find as being repeated elsewhere.
Point 7: ideas in sentence on lines 641 - 643 has already been mentioned previously
Response 7: this part of the text was removed as it is mentioned in the part 3.1 Light
Point 8: ideas in sentence on lines 649 - 651 has already been mentioned previously
Response 8: This text was merged with te text in part 3.4 Culture turbulence (p. 12) as to remove repeated information.
Point 9: ideas in sentence on lines 653 - 658 has already been mentioned previously
Response 9: the texts were revised in order to remove repeated information; now the pertinent text concerning cell density is shown in the part 3.7 Cell Density and Culture Depth (p. 15-16)
Specific comments
Point 10: the concept of alpha slope is introduced quite abruptly here before explanation of the photosynthetic light response curve (Fig. 4) has been introduced. Perhaps refrain from mentioning concepts that are explained only much later in the document.
Response 10: Figure 4 was moved close to the part Photochemical efficiency (pp. 8-9) in which he explanation of alpha and other variables is described.
Point 11: The Mehler reaction is mentioned a few times during the document; perhaps provide a short description when it is first introduced.
Response 11: In the part 2.2 The Dark Reactions the mechanism of the Mehler reaction is briefly described (pp. 5-6) when it is firstly mentioned.
Point 12: could not find a Section 1.2.2 as referenced in line 127
Response 12: It was a mistake; should be Figure 4; the text was moved close to Figure 4 into the part Photochemical Efficiency (p. 9, lines 359-366).
Point 13: what does “the other cycle” refer to in line 241?
Response 13: the sentence was revised to: “In dense microalgae cultures used in microalgae biotechnology, the other, much faster cell cycling is imposed by culture turbulence in the culture layer, which mainly results in intermittent light–dark regime alternating in seconds or milliseconds as compared to the hours or months for diurnal and seasonal cycles [ref].” (section 3 Variables Affecting Photosynthesis and Growth of Microalgae Cultures, page 6, 2nd paragraph)
Point 14: the influence of pulsed light is introduced in lines 291 - 298 and this concept is referenced a number of times throughout the document. If this phenomenon is explained more thoroughly when it is introduced, it will provide a useful foundation to aid in understanding the applications described later, for example, the effects of turbulence and mixing (e.g. lines 491 - 493 etc.)
Response 14: the pulsed (intermittent, flashing) light effect on photosynthesis is mentioned in section 3.1 Light (pp. 6-7), and then in more detail in section 3.4 Culture Turbulence (p. 12).
Point 15: perhaps just an issue of formatting but equation 5 seems unclear e.g. no occurrence of the bicarbonate ion
Response 15: the equation (5) was revised as bicarbonate HCO3- was no properly formatted (p. 14).
Point 16: it is not clear what the purpose of equation 6 is. Was it used to derive equation 7? if so, a little more detail to show the connection will help the reader
Response: The text was added to explain the relation of equations 6 and 7 (p. 14)
Point 17: it will be very helpful if more detail could be given in this document to briefly explain the methods and differences between rapid fluorescence induction kinetics and the saturating pulse analysis of fluorescence quenching
Response 17: The additional text explaining techniques of measurements of fast fluorescence induction and saturating pulse analysis of Chl fluorescence quenching in more detail was included in the part 5.3 Chlorophyll Fluorescence Monitoring(p.22-23).
Point 18: it is not clear what the symbols like Fv or Fm (e.g. Table 1) refer to
the utility of the manuscript will be enhanced a lot if the pulse-amplitude modulation metrics e.g. those introduced in Table 1 could be related to concepts introduced earlier in the document
Response 18: The basic meaning and role of fluorescence variables FV and FM as measured by PAM fluorimetry as well as fast fluorescence induction kinetics was explained and clarified (p. 22-23).
Minor issues
Point 19: line 204: grammar? “has be considered”
Response 19: the mistake was corrected to “has been considered”.
Point 20: use of italics for genus and species names throughout the document e.g. Chlorella, Chlamydomonas reinhardtii etc.
Response 20: All names of strains throughout the text were put in italics.
Author Response File: Author Response.docx
Reviewer 3 Report
Please see attachment for details.
Comments for author File: Comments.docx
Author Response
Response to comments of Reviewer 3
The authors stated several variables regarding photosynthesis and growth (light, intensity, pH, CO2/O2 exchange, nutrient supply, culture turbulence, light/dark cell cycling, biomass density and culture layer (light path)) that affect microalgae mass cultures.
In general, the paper is well-written and provides useful information but needs some major revisions in order to be ready for publication.
Point 1: Figure 1, the authors need at least to mention some products (lipids, carbohydrates, fatty acids, etc.) from microalgae cultivation.
Response 1: Figure 1 was revised - major biomass products were mentioned.
Point 2: Can the authors comment on the feasibility of cultivation systems since they mentioned the significance of the final product value? Also a fiscal comment is necessary to the final efficiency of the various cultivation systems based on statistical data up to 2021.
Response 2: The section 3.8 Cultivation Unit Design and Culture Productivity including Table 1 was added to the manuscript (pp. 16-17). Table 1 shows the overview of advantages and disadvantages of three basic types of cultivation units which are used for microalgae production. In Table 2 the current industrial-scale biotechnology applications of the microalgae biomass with added value.
Point 3: The authors need to describe and comment for the biosynthesis of photosynthetic apparatus as well as the genes, which are involved in photosynthesis. In general, it is known that almost 120 genes (e.g. light dependent genes, or sugar dependent genes which encodes and regulates photosynthetic proteins) are involved in chloroplast protein synthesis and photosynthesis, so an incorporation of the genomics of the photosynthesis will enhance the part of the paper regarding the process of photosynthesis.
Response 3: This article has been aimed to describe and summarize the photosynthetic variables influencing the growth of microalgae. In our opinion the description of protein biosynthesis and the role of individual genes is rather out of the scope of the topic - Variables Governing Photosynthesis and Growth in Microalgae Mass Cultures. According to our opinion this would fit to another kind of manuscript focused more on molecular biology as our MS is more focused on applied research in biotechnology. Thus, we did not include this matter into the submitted manuscript. Nevertheless, as to follow the reviewer’s recommendation, we have added Section 4 Genetic Improvement of Photosynthetic Reactions where the role of genetic manipulation improvement of major photosynthetic pathways was discussed (pp. 19-20). It concerns the modifications of light-harvesting antenna proteins, some enzymes of carbon-reduction cycle and lipid and carotenoid biosynthesis.
Point 4: Authors need to comment more on the direct effects of light to photosynthesis apparatus effects and final products from microalgae biomass.
Response 4: Here, we have not been sure how to understand this comment. Thus, the section ‘3.1. Light’ was revised as to clarify some parts and put new information and references (p. 7, lines 274-281; p. 8, lines 321-340; p. 9, lines 358-366).
Point 5: Also in temperature and dissolved oxygen more specific details about what changes caused to the photosynthesis system and the final products of microalgae should be mentioned
Response 5: The sections of ‘3.2 Temperature’ and 3.3 Dissolved Oxygen’ were revised and rewritten. In section 3.3 Dissolved oxygen, the last paragraph (p.11-12), the last two paragraphs are aimed to the synergistic effect of temperature, high dissolved oxygen concentration under high irradiance as concerns the overproduction of some photosynthesis products.
Point 6: In the nutrient filed the authors should include more resent specific details of the effects of micro and macronutrients, or C/N ratios in the microalgae growth as well as the final products. Moreover, other elements like aluminum or fluoride may be commented for their effects to growth of microalgae.
Response 6: The section of ‘3.6 Nutrition’ (pp. 15-16) was significantly revised and extended. The C/N ratios were discussed as well as involvement of various elements in microalgae nutrition. For example, in our experiments we use inorganic medium for phototrophic growth which contains (in mg L-1): KNO3, 2021; KH2PO4, 340; MgSO4×7H2O, 989; ferric-sodium chelatonate, 18.4; CaCl2, 16; H3BO3, 1.9; MnCl2.4H2O, 7.3; ZnSO4×7H2O, 7; CuSO4×5H2O, 2; CoSO4×7H2O, 1.4; (NH4)6Mo7O24, 0.08; and NH4VO3, 0.06; pH 7.4 (Zachleder and Šetlík 1982, Biol Plant 24:341-353). As far as we know no aluminium or fluoride salts are added, not even as trace elements to growth medium for microalgae. According to our knowledge these elements would be rather photosynthesis-inhibiting.
Point 7: Recent studies demonstrate also an alternative effort for enhancing photosynthetic CO2 in microalgae by genetic engineering of the Calvin cycle or other like transformation of chloroplasts with Rubisco subunits. Authors can also comment in a separate paragraph some of these studies as alternatives for photosynthesis manipulation.
Response 7: As in Response 3 to follow the reviewer’s recommendation, we have added Section 4 Genetic Improvement of Photosynthetic Reactions where the role of genetic manipulation improvement of major photosynthetic mechanisms was discussed (pp. 19-20).
Author Response File: Author Response.docx
Reviewer 4 Report
see attached file
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 4 Comments
Minor Corrections
Point 1: Line 19: Replace “culture layer” not “culture depth”
Point 2: Line 77: Replace “layer” with “depth” also Lines 255, 267
Response 1-2: Points 1-2 were corrected as suggested by the reviewer.
Point 3: Lines 85 - 87: Poorly worded sentence, light is not converted directly to biomass
Line 87: Replace “microalgae about” with ”cyanobacteria at least”
Response 3: The sentence lines 85-86 was changed to: “The process emerged in cyanobacteria at least 2.5 billion years ago.”
Point 4: Line 105: Replace “NADP H2” with “NADPH2” also Line 145
Response 3: Throughout the text “NADPH2” is used.
Point 5: Line 121: Replace “the” with “a”.
Response 5: It was corrected.
Point 6: Figure 3: Stoichiometry of the Calvin-Benson cycle is not made clear.
Response 6: The stoichiometry and the partial reactions in the diagram were revised using some recent references.
Point 8: Line 197: Reference required
Response 8: Reference was added.
Points 9-39: mostly corrections of words or terms were required
Point 9: Lines 227-228: Reword as “In contrast, “long time scale” responses are ecological and evolutionary adaptations….”
Point 10: Line 243: Insert “for diurnal and seasonal cycles” after “months”
Point 11: Lines 246-247: Replace “is carried” with “takes place”
Point 12: Line 284: Replace ”higher” with “greater”
Point 13: Line 292: Italics for “Chlorella” also Line 327, Line 416, Line 784, Line 864, Line 948
Point 14: Line 299: Replace “precious” with “precise”
Point 15: Line 311: Replace “source” with ”sources”
Point 16: Line 323: Replace “in” with “with”
Point 17: Line 330: Italics for “Chlamydomonas reinhardtii”
Point 18: Line 331: Replace “s in” with “under”
Point 19: Line 421: Insert “they” before “are”
Point 20: Line 429: Replace “constrains” with “constraints”
Point 21: Line 448: Replace “reaction” with “reactions”
Point 22: Line 449: Replace “ability” with “rate”
Point 23: Line 455: Italics for “Nannochloropsis”
Point 24: Line 458: Insert “most” before “suitable”
Point 25: Line 513: Replace “other” with “others”
Point 26: Line 525: Replace “flagellae” with “flagella”
Point 27: Line 525: Italics for “Dunaliella”
Point 28: Line 595: Replace “25times” with “25 times”
Point 29: Line 607: Replace “amount is” with “amounts are”
Point 30: Line 679: Delete “the” before “volumetric”
Point 31: Line 759: Replace “and” with “with”
Point 32: Line 781: Italics for “Haematococcus”
Point 33: Line 782: Italics for “Arthrospira”
Point 34: Line 784: Italics for “Monoraphidium”
Point 35: Line 794: Delete “us the”
Point 36: Line 812: Replace “ways” with “pathways”
Point 37: Line 816: Replace “F0” with “F0”
Point 38: Line 865: Replace “L-1” with”L-1”
Point 39: Line 952: Italics for “Chlorella fusca”
Responses 9-39: have been revised as suggested by the reviewer.
Questions/Comments
Point 40: Lines 530-532: How easily are the thin layer cultures scaled up?
Response 40: The scale-up of thin-layer systems is technically more complicated and requires higher costs as compared with raceway ponds; larger area is also required. Nevertheless, large facilities have been built, for example 3,000 m2 units cascade raceways were constructed in Portugal and some more are planned in Spain.
Point 41: Lines 746-749: Could give a little more detail on the protocol used.
Response 41: More detailed description of the respirometry was added (p. 24, section 5.2 Measurements of oxygen evolution, end of the last paragraph) - “The methodology allows to distinguish between the metabolisms of the three main populations: the microalgae, the heterotrophic bacteria and the nitrifying bacteria. Firstly, samples of the microalgae cultures were taken and subjected to nutrient starvation to remove the organic matter and the ammonium present in the medium. Subsequently, the samples are placed inside the jacketed flask and the variation in dissolved oxygen over time was measured during four light–dark periods of 4 min when the variation in dissolved oxygen over time was measured and registered.”
Point 42: Line 931: Need to briefly explain FTIR technique. More detailed description of FTIR spectroscopy was added.
Response 42: One sentence has been added in the part 4.4: “Using Fourier Transform Infrared Spectroscopy (FTIR) carbon allocation analysis can be performed which allows the quantification of the relative amounts of major biomass components [ref] (pp. 18-19).”
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors replied to the comments.
