Review Reports

Round 1

Reviewer 1 Report

Review on: Applied Sciences, Manuscript ID: applsci-1144380

Title: Assessment of the effect of light and temperature fluctuations on desert algae growth and metabolism processes

 

The study investigated the effect of dynamic light and temperature conditions on growth performance, pigments concentration, and macromolecular composition in an alga isolated from the Qatar desert (Nannochloropsis sp.). The data were compared to growth conditions of constant light and temperature to evaluate the potential to acclimate to such fluctuating conditions. The authors found different impact of temperature versus light fluctuations. In the condition of combined temperature and light dynamics, the fluctuation of irradiance exerted strong (negative) impact on algal physiology and growth performance. The authors draw conclusions on how to design outdoor cultivation systems to avoid the negative outcome of dynamic environmental conditions.

First of all, the research topic is interesting and deserves attention. However, in its present state the study is relatively far from being ready for acceptance. This is due to a number of omissions and even flaws in the methodological procedure. Quite a number of methods were not decribed in the methods section. The attempt to measure Chlorophyll a and b in an algal strain known to possess Chl a only (!) documents a serious gap of biological expertise. It also means that the data on Chl concentrations were not measured correctly.

The use of specific terms and phrases is quite often not appropriate and leaves the reader with the impression of a lack of expertise by the authors.

English language needs strong improvement. Some of the species mentioned in the introduction are not given in full name.

The investigated strain was isolated from the Qatar desert and is described as ‘promising for outdoor produtcion’. Therefore, I would expect much more information on this strain to justify this statement and not just a reference.

The results section contains already too much discussion of experimental data whereas the discussion section adds very little contribution to a deeper understanding of the results. In fact, only the last paragraph of this section could be assigned adequate for a discussion. Moreover, to my opinion the authors have drawn conclusion that do not fully meet the experimental data. As an example, the data clearly show that temperature fluctuations do even improve biomass productivity, the authors however suggest to keep the temperature constant during outdoor cultivation. They further speculate on growth performance in the winter but there are no data in this study to support such speculation. Light fluctuations on the other hand showed a clear negative impact on productivity however, the authors claim that it is not possible to influence irradiance levels outdoors. To my opinion, this is not entirely true since the layer depth of the cultivation system will have strong impact on the average irradiance level of the culture. The authors also state that the investigated algal strain showed a high level of adaptation to light and temperature stress. First of all, the authors should make themself aware about the difference between ‘adaptation’ and ‘acclimation’. Second, the level of adaptation was not measured or refered to a specific result. Just ‘surviving’ is not a measure of the level of acclimation. Moreover, it would be necessary to compare such level of acclimation to e.g. other species. And third, I do not agree to this statement since the alga showed this severe decrease of productivity under irradiance fluctuations. To me, this is rather the opposite of a high level of acclimation to these conditions and is a quite surprising result for an algal strain isolated from the desert.

To me it would make much more sense to first evaluate the temperature and irradiance optimum of this strain and to then prove this under dynamic conditions representative for the entire seasonal change including winter season.

In general, the text is highly repetitive and needs to be edited in this respect.

In the following I will give some more specific comments but they do not cover all points of criticism:

- title line 3: only one alga was investigated

- line 11: the authors investigated the ‘macromolecular composition’ but not the ‘structure of cell components’

- line 16: it is not clear why 150 µmol photons m-2 s-1 are the minimum irradiance under summer conditions?

- line 22: ‘growth rate’ or ‘biomass productivity’ but not ‘growth productivity’

- line 26: ‘both light and temperature effects were found related’ this is a very vague statement

- line 48: ‘light and temperature are the most important factors for better algal growth’ - this is not true since nutrient deprivation can strongly override any of these factors!

- line 97: it is stated that Nannochloropsis sp. QU130 contains 9% lipids. This is certainly too low for biofuel production as suggested here.

- line 101: what is ‘robustness’?

- line 115: what is ‘fully controlled’?

- line 119: ‘fully controlled, adjustable and automated’ - isn’t it all the same?

- line 146: ‘culture media was continuously adjusted to avoid mineral limitation’ what does that mean exactly?

- lines 150 – 153: completely unclear!

- line 154: ‘ three consecutive days’ this is not the sampling procedure as shown in Fig. 3

- line 202: ‘algae biomass was freeze-dried’ how exactly? Did the authors wash the samples to remove salts from the medium?

- line 210: this equation is not appropriate for samples containing Chl a only

- line 314: measurement of cell size is not described

- line 327: ‘clarified by the orientation of cells to the division process’ – what does this mean?

- line 333: ‘low light’ ‘low temperature’ - it is defined what ‘low’ exactly means here (below optimum?).

- line 349: measurement of carotenoids is not described

- Fig. 7: it is not clear how the x-axis relates to the x-axis in Fig. 4. It is not possible to figure out at which time point the conditions were changed.

- line 372: the authors did not measure a ‘metabolite profile’ !!

- line 373: it is not clear what is shown in Fig. 8 – is it the mean value of the four time points?

- line 391: ‘analysis of metabolites production’ - lipids are missing in Fig. 9!

- Fig. 9: the values for standard deviation seem to be erronuous and random. In Fig. 9A there are no SD values for total sugars. To my opinion, SD values are far too low with respect to the used procedure of quantification. I do not trust these data.

- line 421: ‘increase in biomass concentration with increase of light intensity’ – this is trivial!

- line 430: ‘by the reduction of proteins’ – proteins are usually not employed for cellular energy gain!

- line 439: ’70 tons of biomass per hectare’ – I do not think that it is feasible to calculate an annual outdoor production from an experimental setup in a closed bioreactor and for summer conditions only!

- line 441: authors state that control of temperature is not realistic under desert conditions but the suggest this control few lines later (line 481).

 

Author Response

Please see the attachment.

Author Response File: Author Response.doc

Reviewer 2 Report

I apologize for being late with this review.

I read the abstract and the Figures + Captions to provide some feedback which I hope is useful to the authors.

The authors grew a model microalga Nannochloropsis under a matrix of light and temperature regimes including ecophysiologically reasonable models of conditions in the summer Qatari desert.  It is useful to study responses under reasonable conditions.

The authors conducted compositional analyses of the microalgae under these different conditions including total biomass, protein, lipid, total sugar, chl a and carotenoids.

The presentation of the compositional data is scattered across 9 figures with arbitrary and inconsistent formatting.

I recommend the authors consolidate their results to fewer figures that clearly present their compositional information in the context of the relevant light and temperature regimes.

There are multiple figures showing only sub-sets of the light and temperature regimes which could be more appropriately replaced with backgrounds underlying actual data presentation in fewer, graphically consistent figures.

The English expression is comprehensible but filled with errors of expression, and would benefit from thorough language revision.

best regards, in haste, Doug Campbell

Abstract:
At night umol photons m-2 s-1 goes to ~0.

"...when those factors reach their maximum rates..."
I think:
"...when temperature and light fluctuations are widest..."

Nannochloropsis should be italicized.

The abstract is quite long, but perhaps this is the journal style?

"The strong effect of dynamic conditions was revealed, where both light and temperature effects 26 were found related"

rather
"Dynamic light and temperature showed interactive effects ."

The English is comprehensible but filled with minor errors of expression and idiom;  I am sorry I lack the time to do the English proof reading.

Figures:
Figure 2 should include light and temperature profiles on all panels (straight lines for constant light and temperature)

Figure 4:
Y axis labels are difficult to understand; what does the 'X' mean?

This figure would be much easier to understand if overlaid with traces of the light and temperature regimes.

Panel A covers Experiment C.
Panel B covers Experiment B.
Panel C covers Experiment D.

Why erect multiple levels of opaque labelling coding which is inconsistent across panel labels and experiment labels?

Figure 5: Again, what is the meaning of the 'X'?
I have never seen that format for an axis label before.

Figure 5 panel D suddenly switches to a filled area for Temperature (I think) instead of connect-the-dots lines in other panels.

Figure 6 same comments.

Figure 7
Figure + Legend + Caption should be an interpretable unit.
What is the numeric scale for 'stress factor'?
Probably explained in the text, but not clear from the Figure + Legend + Caption.

Figure 8
Bars for total sugar, points with connect the dots for Protein and Lipids.  Not valid to connect the dots across a categorical X axis.

Figure 9
Same layout as Figure 6, but here the metabolite determinations are not connected with lines.

Slightly odd that total lipids are not presented when in many cases they are the main goal for aquaculture production.

 

Author Response

Please see the attachment.

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

The authors submitted a revised version of the manuscript with some changes according to the suggestions in the review of the first manuscript version. Although some points are clearer now, there are still a number of major issues that were not addressed in the revised version.

First of all, the equation for the calculation of Chlorophyll a content (and of course also the resulting data) is wrong here! I stated that clearly in the first review but obviously the authors did not understood the background of these equations. The authors are asked to check the literature to solve this problem for future submissions.

Second, the authors are still using phrases that are not appropriate and reveal a lack of biological expertise, e.g. the use of the term ‘metabolite’ and ‘primary metabolite’. The statement that ‘lipids, carbohydrates, proteins and pigments are primary metabolites’ is completely wrong. Consequently, the authors did NOT analyze the metabolite profile.

Third, I still do not agree with the major conclusion that Nannochloris QU130 is well-adapted to a combination of temperature and light fluctuations as applied here. The strong accumulation of lipids in combination with the lowered growth rate under these conditions is a clear indication of an elevated stress level. This is even more surprising since the temperature range fluctuates around the optimal temperature and the light fluctuations itself did not induce an increased stress level. This is not addressed by the authors at all.

Fourth, whereas the introduction focuses on the summer conditions in the Quatar desert, the results are discussed with respect to year-round conditions. To my opinion, the experimental design of the study does not support such conclusions. So, if the aim of the study was to investigate the potential of this algal strain for outdoor and year-round biomass production then this aim was not fulfilled. On the other hand, if the study remains on the level of investigating summer conditions I am afraid it does provide significant scientific input.

Based on these major points I do still not recommend publication.

Author Response

Please see the attachment

Author Response File: Author Response.doc

Reviewer 2 Report

'X' as a symbol/abbreviation for Algal dry weight concentration is an odd, and opaque choice.

In Figure 4 the 'X' appears both inside, and outside, the (units).

In Figure 5 the units symbols vary erratically from panel to panel, for ?no? reason; ex. (kg;m-3) vs. (kg.m-3)

Figure 5 uses odd 'shading' on some graphic elements. Just because Excel can do it, does not make it a good idea.

Figure 6. 'N (Cells.ml-1)';   What is 'N'?

As I wrote in my previous review, Figure + Legend + Caption should be a comprehensible unit, on their own.  Using undefined, obscure abbreviations does not help the reader.

Graphic elements in Figure 6. also overlay each other; 'Trial 3' overlaps with the right Y axis labels (at least on my screen).

Figure 7: 'Stress Factor'.  Easy enough to define in the caption as A480/A665.

 

Conclusions:

'

Light cycles with constant temperature demonstrated a decrease of 50% in biomass 444 concentration following an acclimation time of around 5 days.'

compared to what? Percent change always has to be expressed relative to a baseline.

The relevant paragraph is backwards; why put the % change first, and then explain what it means in the following sentences?

 

"

Under the temperature fluctuation regime with constant light, a decrease of 15% in 449 growth productivity was first observed,"

Again, compared to what? % change is only meaningful as an explicit comparison to a baseline.  I would just give the key numbers and leave out the confusing % changes.

 

Author Response

Please see the attachment

Author Response File: Author Response.doc

Round 3

Reviewer 1 Report

The second revised version of the manuscript clarifies a number of issues. It is now clear for the reader that the focus is on the ‘summer condition’ and that this condition is predominant throughout the year. This information helps a lot to judge the informations/data in the manuscript.

The issue with the determination of the Chlorophyll content is still not solved adequately. The critical point is that the equations for the estimation of Chl a/b contents of plant material have been established empirically and they are valid only in a restricted range of Chl a/b ratios. This is due to the fact that there is an overlapping absorption of Chl a and b in the red spectral region. This means that such an equation MUST NOT be used for the determination of Chl a in a species containing Chl a only. The absorption value at 650 nm would take into account the overlapping absorption by Chl b. However, when there is no Chl b present the substraction of this absorption value (as done in the equation of the present manuscript) leads to an underestimation of the Chl a content! The authors have to use the Chl a-specific absorption coefficient for a specific solvent as provided by e.g. Porra et al. (BBA 1989, 384-394) to calculate the correct Chl a content. Although the Chl a content is not the most important parameter in this manuscript it is no good scientific practice to publish such an inadequate equation.

There are some minor points and I have to apologize to not have mentioned them before:

- units should be given in a consistent format; currently, there are two formats: e.g. kg.m-3 and g m-2; in addition: there should be a space between value and unit – this is not always the case.

- line 209: the unit  (g L−3  d−1) does not make sense; either it is (g L−1  d−1) or (g m−3  d−1)

- Figure 2: subfigures C and D should be changed in order of appearance (C on the left, D on the right); subfigure C: x-axis should have the same scaling as the other subfigures

- Figure 7: A480/A665 is not explained in the figure legend; despite the formal issue: this so-called stress factor is not explained (what kind of stress is indicated by this factor?), it is hardly mentioned in the results and not discussed at all. To me, this factor does not add any relevant information in the present manuscript.

- the term ‘sugar’ is usually employed for soluble carbohydrates. Is this really intended by the authors here or is it rather the total carbohydrate (including e.g. starch) content?

Author Response

Please see the attachment.

Author Response File: Author Response.doc