SS symmetry
Article
The Microalga Chlorella vulgaris as a Natural Bioenergetic
System for Effective CO2 Mitigation—New Perspectives against
Global Warming
Fanourios Mountourakis, Aikaterini Papazi and Kiriakos Kotzabasis *






Citation: Mountourakis, F.; Papazi,
A.; Kotzabasis, K. The Microalga
Chlorella vulgaris as a Natural
Bioenergetic System for Effective CO2
Mitigation—New Perspectives
against Global Warming. Symmetry
2021, 13, 997. https://doi.org/
10.3390/sym13060997
Academic Editor:
Eleftherios Touloupakis
Received: 9 May 2021
Accepted: 1 June 2021
Published: 2 June 2021
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Symmetry 2021, 13, 997. https://doi.org/10.3390/sym13060997
Department of Biology, University of Crete, Voutes University Campus, 70013 Heraklion, Greece;
grad937@edu.biology.uoc.gr (F.M.); psipsinel80@yahoo.gr (A.P.)
* Correspondence: kotzab@uoc.gr; Tel.: +30-2810-394059; Fax: +30-2810-394408
Abstract: In the present contribution, the differentiation in the molecular structure and function of
the photosynthetic apparatus of the unicellular green alga Chlorella vulgaris was studied at several
light intensities (0–400 µmol m−2 s−1 ) and various CO2 concentrations (0.04–60% CO2 ), in completely
autotrophic conditions. Asymmetries that occur by different light intensities and CO2 concentrations
induce metabolic and functional changes. Using chlorophyll fluorescence induction techniques
(OJIP test), we showed that Chlorella vulgaris tolerates extremely high CO2 levels and converts them
photosynthetically into valuable products, including O2 and biomass rich in carbohydrates and lipids.
Interestingly, the microalga Chlorella vulgaris under extremely high CO2 concentrations induces a
new metabolic state intensifying its photosynthetic activity. This leads to a new functional symmetry.
The results highlight a potent CO2 bio-fixation mechanism of Chlorella vulgaris that captures up to
288 L CO2 L PCV−1 day−1 under optimal conditions, therefore, this microalga can be used for direct
biological CO2 -reducing strategies and other green biotechnological applications. All of the above
suggest that Chlorella vulgaris is one of the most prominent competitors for a closed algae-powered
bioreactor that is able to consume huge amounts of CO2 . Thus, it is a sustainable and natural
bioenergetic system with perspectives in dealing with major environmental issues such as global
warming. In addition, Chlorella vulgaris cultures could also be used as bioregeneration systems in
extraterrestrial missions for continuous atmospheric recycling of the human settlements, paving the
way for astrobiological applications.
Keywords: Chlorella vulgaris; CO2 mitigation; global warming; biofuels; photosynthesis; environmen-
tal biotechnology; astrobiology
1. Introduction
Over the last couple of centuries, industrialization along with urbanization have
brought an immense increase in greenhouse gases (GHGs), mostly carbon dioxide (CO2 ),
which is considered one of the main causes of global warming [1] and other major environ-
mental problems, such as ocean acidification [2]. Atmospheric CO2 concentration increased
rapidly from 280 ppm in 1850 [3] to more than 417 ppm in 2021, with more than half of this
increase happening in the last 30 years [4]. Notably, CO2 levels today are higher than at any
other point in at least the past 800,000 years [5]. Human activities release about 35 gigatons
of CO2 every year, as opposed to almost 10 megatons 2 centuries ago [6]. There is already
clear evidence that anthropogenic emissions of GHGs, such as CO2 , alter the natural carbon
cycle, which leads to an accelerated warming of our planet [7]. Based on Representative
Concentration Pathways (RCPs), adopted by the International Panel on Climate Change
(IPCC), and other modelling scenarios, scientists predict that the amount of CO2 in the
atmosphere might double or even triple at the end of the 21st century [8,9]. This will result
in a planet that is hotter by more than 4 ◦ C compared to preindustrial ages [8,9], well above
the 2 ◦ C danger mark agreed in COP21 Paris Agreement [10].
https://www.mdpi.com/journal/symmetry
Symmetry 2021, 13, 997 2 of 16

Several techniques have been examined worldwide for reducing CO2 emission levels
based on chemical, physical and biological methods [11–13]; among these, CO2 bio-fixation
via the photosynthetic procedure is considered one of the most effective approaches for
CO2 capture [14,15]. Photosynthesis is the process used by phototrophic organisms to
convert light into chemical energy that is then invested in carbon fixation, the conversion
of inorganic carbon compounds (usually CO2 ) into organic carbon compounds (usually
a carbohydrate such as glucose) [16]. Oxygenic photosynthesis, the mechanism that uses
H2 O as a reducing agent and releases O2 , evolved 3.8 × 109 years ago and changed Earth’s
atmosphere [17]. It slowly converted a strongly reducing, CO2 rich, atmosphere into the
oxidizing one that we know today [18]. Undoubtedly, photosynthetic organisms have a
fundamental regulatory role in the natural carbon cycle equilibrium [19], an attribute in
which we should invest while tackling the spiraling problem of CO2 increase.
Regulations and controls on reducing our “carbon footprint” cannot help us much
without serious socioeconomic impacts on existing infrastructure [1]. However, an efficient
photosynthetic system, such as microalgae, could simultaneously be profitable through
numerous applications, while providing a green solution for CO2 mitigation. Microalgae
are the fastest growing photosynthetic organisms on earth, up to 50 times faster than
their terrestrial plants [20]. They do not require cultivable land, while their use of fresh
water is dramatically reduced [21]. Various microalgae tolerate a wide range of cultivation
conditions [22], including extremely high CO2 concentrations [23,24]. In particualr, Chlorella
vulgaris photoautotrophic cultures cultivated in bubble column photobioreactors showed
a high rate of CO2 fixation up to 10% [25,26]. Comparatively, photosynthetic activity in
most terrestrial plants increases when CO2 levels rise, until some saturating concentration,
which is typically around 0.1%. Higher CO2 concentrations lead to adverse effects [27].
The microalga Chlorella vulgaris is an extremely promising candidate also for large-
scale cultivation that has already attracted a lot of interest for numerous applications [28].
A rapid growth rate and a resistance to harsh environmental conditions are some of the
features that make this microalga a prominent candidate for efficient production of high-
value biomass yields [29]. Due to its high nutritional value, it is one of the few microalgae
that have been used successfully as alternative aquacultures, and a source of animal and
even human food [30–32], as it exhibits a large number of therapeutic properties widely
used in pharmaceutical and cosmetic industry [29]. Furthermore, agrochemical applications
of Chlorella vulgaris can benefit common crops by both biofertilization [33] and growth
acceleration [34]. Another interesting feature of this particular species is that it responds to
different growth parameters by modifying its rich biochemical composition [28], which is
ideal for third-generation and even fourth-generation biofuels, that are not involved in the
“food vs. fuel dilemma” [35]. Both bioethanol and biodiesel production can be optimized
by aiming at carbohydrate and lipid accumulation, respectively [36,37].
In the present contribution, the metabolic and functional differentiation of the uni-
cellular green alga Chlorella vulgaris was studied at various light intensities and CO2
concentrations, aiming at effective CO2 mitigation, conversion of CO2 to O2 and the pro-
duction of biomass rich in carbohydrates and lipids. Such a microalgal system could be the
key for environmental and astrobiological applications.

2. Materials and Methods

2.1. Organism and Cultivation Conditions
Cultures of the wild type strain of the unicellular green alga Chlorella vulgaris (211-11b,
SAG Culture Collection of Algae) [38] were used for this study. According to Wong et al.
(2017), Bold’s Basal Medium (pH = 6.8 ± 0.1) was found to be the best liquid medium for
biomass production in Chlorella vulgaris [39,40]. The cultures were grown in elongated
glass tubes (Ø 5 cm × 50 cm), and incubated at controlled temperature (25–26 ◦ C) [41] with
continuous sterile air bubbling (50–60 L·h−1 ), for about a week. Light was provided with
warm white LED lamps with the intensity of 40–50 µmol m−2 s−1 and a photoperiod of 16 h
light:8 h dark [42]. After reaching a sufficient amount of biomass with high photosynthetic
Symmetry 2021, 13, 997 3 of 16

efficiency, the cultured cells were centrifuged at 1500× g for 5 min. Subcultures with an
initial concentration of 1 µL packed cell volume (PCV) per mL culture were initiated with
new medium. The initial cell density of 1 µL PCV mL−1 culture was the best starting
point so that all the photosynthetically active cells absorb about the same amount of
light, while avoiding fast nutrient depletion. All the experiments were carried out in
120 mL hermetically closed bottles (Ø 5 cm, height 9.5 cm) with rubber septa and in
the absence of any organic carbon source. The final culture volume in each bottle was
50 mL autotrophic culture medium (liquid phase) and 70 mL gas phase with different
CO2 concentrations (0.04–60%). Increased CO2 levels decreased the pH of the medium
from 6.8 (without CO2 addition) down to 5.2 (in treatments with 60% CO2 ), which was
then increased proportionately to the photosynthetic activity of the microalgae [43]. The
bottles were kept in a temperature-controlled chamber (25–26 ◦ C) at various light intensities
(0–400 µmol m−2 s−1 ). Sampling took place at the same time (at the middle of the light
period), using sterile gas tight needles, without opening the bottles.

2.2. Microalgae Growth Determination

The culture’s growth rate was estimated by measuring the packed cell volume (PCV)
of the culture according to the method of Navakoudis et al. [44]. Briefly, a 1 mL sample
of a homogenized cell suspension was centrifuged at 1500× g for 5 min using graduated
capillary hematocrit tubes (TPP, Sigma-Aldrich, St. Louis, MO, USA) and the cell volume
was expressed as µL PCV mL−1 .

2.3. GC-TCD Measurements

Oxygen and nitrogen measurements were made utilizing gas chromatography, using
a thermal conductivity detector (GC-TCD) (Shimadzu GC 2010 Plus, Kyoto, Japan). To
separate O2 and N2 , argon was used as the carrier gas under the pressure of 5 bars, and
at an oven temperature of 120 ◦ C. The column used was a capillary Vici Metronics MC
(Poulsbo, WA, USA) with length of 30 m (diameter 0.53 mm) and film thickness of 20 µm.
The temperature of TCD was set at 200 ◦ C for the detector and 180 ◦ C for the injector. A
gas-tight syringe (250 mL) was used for sampling from the hermetically closed bottles. The
quantification of all gases was carried out by injecting known quantities of O2 and N2 in
the GC-TCD.
For the CO2 measurements, helium was used as the carrier gas under pressure of
five bars and at oven temperature of 250 ◦ C. The column used was the same as explained
above. The temperature of TCD was set at 300 ◦ C for the detector and 280 ◦ C for the
injector. The quantification of CO2 was carried out by injecting known quantities in the GC-
TCD. Preliminary experiments with microalgal cultures in high CO2 concentrations and
in closed cultivation systems confirmed that the decrease in CO2 volume (photosynthetic
CO2 fixation) was about the same with the increase in O2 volume (photosynthetic O2
production) (data not shown).

2.4. Photosynthetic and Respiratory Activity Measurements

For the measurements of photosynthetic and respiratory activity of the cultivated
microalgae, we used GC-TCD measurements (for details, see above). The changes in O2
levels in Chlorella vulgaris closed cultivation systems (initial cell concentration 1 µL PCV
mL−1 ) was measured at known time intervals under light conditions and in absolute
darkness (for the first 16 h of incubation in light and in continuous darkness respectively)
for the determination of the photosynthetic and respiratory activity respectively. The
photosynthetic activity represents the actual net microalgal photosynthetic rate in the corre-
sponding light intensity and CO2 concentration used in each experimental treatment. Each
net photosynthetic value was calculated by the difference between gross photosynthesis
(total O2 production) and the O2 consumption due to respiration. The photosynthetic and
respiratory activity was expressed as µL O2 µL PCV−1 h−1 .
Symmetry 2021, 13, 997 4 of 16

2.5. Fluorescence Induction Measurements: OJIP-Test

The Handy Plant Efficiency Analyser, PEA (Hansatech Instruments, Kings’s Lynn,
Norfolk, UK) was used for the fluorescence induction measurements [45]. This protocol
is based on the measurement of a fast fluorescence transient with a 10 µs resolution in a
time span of 40 µs to 1 s. Fluorescence was measured at a 12-bit resolution and excited by
three red light-emitting diodes providing a saturating light intensity of 3000 µmol m−2 s−1 .
For the fluorescence induction measurements, we put the flat bottom of the small culture
bottle (Ø 5 cm) directly on the PEA sensor using a specific adaptor that ensures absolute
darkness for at least 10 min (in order to open the PSII reaction centers) before the exposure
to saturated light [43]. Under these conditions, and without opening the culture bottles, we
measured the photosynthetic efficiency (Fv/Fm) of the culture in the ambient conditions,
according to the OJIP method [45,46]. Out of all the other photosynthetic OJIP variables
measured, we used the density of active photosynthetic reaction centers (RC/CS0 ), the
functional antenna size (ABS/RC), the primary photochemistry (PSI0 ), the dissipated
energy flux per active reaction center (DI0 /RC), the performance index on absorption basis
(PIabs ) and the maximum photosynthetic efficiency (Fv/Fm) [47].

2.6. Lipid Extraction and Quantification

The protocol of Folch and Stanley [48] was used for the extraction of total lipids,
as discussed by Sati et al. [49], with some slight modifications. Briefly, 3 mL of chloro-
form:methanol in a ratio 2:1 (v/v) were added in 2.5 µL PCV pellet of centrifuged microalgal
cells (1500× g for 5 min) and incubated for 15–20 min with frequent vortexing. The samples
were mixed with 1 mL of 0.74% NaCl solution for two phase separation and centrifuged
for 5 min at 1500× g. The lower phase (chloroform with dissolved lipids) was isolated and
incubated at 95 ◦ C for complete chloroform evaporation [48,49].
For the total lipids quantification, the method of Park and Jeong [50] was used with
slight modifications. Briefly, the lipids were dissolved in 200 µL of H2 SO4 (99,99%), by
vortexing briefly. The samples were then incubated at 95 ◦ C for 10 min and immediately
cooled at room temperature. 4 mL of phosphor-vanillin reagent (500 mL H3 PO4 85%,
125 mL ddH2 O and 0.75 g Vanillin) was added into the samples and incubated for an
additional 10 min at room temperature, before being measured spectrophotometrically at
530 nm. The quantification of total lipids was estimated using a calibration curve with
canola oil.

2.7. Carbohydrate Extraction and Quantification

For the extraction and quantification of total carbohydrates, the method of Schulze
et al. [51] was used. Briefly, 1 mL of HCl 37% was added to 2.5 µL PCV of centrifuged
microalgal cells (1500× g for 5 min) and incubated for 15 min. Samples were then diluted
with 5 mL of ddH2 O. At 400 µL of the diluted samples, we added 900 µL of thymol-
sulfuric acid reagent (0.1 g thymol into 100 mL of H2 SO4 99.99%), and vortexed briefly.
The samples were then incubated for 30 min at 95 ◦ C. The absorbance was measured at
505 nm. The quantification of total carbohydrates was estimated using a calibration curve
with glucose [51].

2.8. Data Analysis

Each experiment was repeated at least three times, thus, each treatment included three
independent samples. Standard errors of the average values are presented on all diagrams.
ANOVA One Way and Tukey HSD paired tests were used for testing the significance of
the values. The normality of samples was tested using the Shapiro–Wilk test and the
homoscedacity using the Bartlett’s test.
 2.8. Data Analysis
Each experiment was repeated at least three times, thus, each treatment included three
independent samples. Standard errors of the average values are presented on all diagrams.
Symmetry 2021, 13, 997 ANOVA One Way and Tukey HSD paired tests were used for testing the significance of 5 ofthe
16
values. The normality of samples was tested using the Shapiro–Wilk test and the homosce-
dacity using the Bartlett’s test.
3. Results
3. Results
3.1. Effect of Different Light Intensities on the Microalgal Photosynthetic Mechanism
3.1. Effect of Different Light Intensities on the Microalgal Photosynthetic Mechanism
The effects of various light intensities (0–400 µmol m−2 s−1 ) on the photosynthetic
Τhe effects of various light intensities (0–400 μmol m −2 s−1) on the photosynthetic appa-
apparatus of Chlorella vulgaris under natural atmospheric conditions in closed cultivation
ratus
systems of Chlorella vulgaris under
were evaluated. natural atmospheric
The kinetics of the maximal conditions in closed efficiency
photosynthetic cultivation(Fv/Fm)
systems
were evaluated. The kinetics of the maximal photosynthetic efficiency
in Figure 1A showed that increased light intensity acts as a major stress factor for the (Fv/Fm) in Figure 1A
showed that increased light intensity acts as a major stress factor
photosynthetic mechanism under ambient CO2 (0.04%) concentration. In complete dark- for the photosynthetic mech-
anism
ness (0under m−2 s−CO
µmolambient 2 (0.04%)remained
1 ), Fv/Fm concentration. In complete
constantly high.darkness (0 μmolofmdays,
After a couple −2 s−1), Fv/Fm
under
remained constantly high. After a couple of −
days,
2 −under
1
extremely low light conditions (10 µmol m s ), Fv/Fm values showed a slight de-extremely low light conditions (10
μmol
crease.mAss light
−2 −1), Fv/Fm values
intensity showed to
increased a slight
50 µmol − 2
decrease. − 1
m s As, light intensity increased
the photosynthetic to 50 μmol
efficiency drop
m −2 s−1, the photosynthetic efficiency drop came earlier and sharper, leading to a decrease in
came earlier and sharper, leading to a decrease in photosynthetic capacity. Stepping up
to 100 µmol m−capacity.
photosynthetic 2 s−1 and Stepping
200 µmol upmto−2100
s−1μmol
, Fv/Fmm−2 decreased
s−1 and 200down μmol to m−20.4
s−1(Figure
, Fv/Fm1A). de-
creased downatto400
Specifically, 0.4 µmol
(Figure −2 s−
m1A). 1 under ambient
Specifically, at 400 μmol
CO2 , m −2 s−1 under ambient CO2, there was
there was a total breakdown of the
aphotosynthetic
total breakdown of the photosynthetic
apparatus after the fourth apparatus after day
incubation the fourth incubation
(no reliable day (no reliable
measurements with
measurements
OJIP-test; no observed with OJIP-test;
bulletsno inobserved bullets
Figure 1A). in Figure
These 1A). These
first results showed firstan
results showed
extremely an
light
extremely light sensitive photosynthetic
sensitive photosynthetic mechanism under mechanism
ambient under
carbonambient
dioxide carbon
withdioxide
limitedwith lim-
biomass
increase
ited biomass (Figure 1B). (Figure 1B).
increase

Figure 1. (A): Kinetics of the microalgal photosynthetic efficiency, expressed in Fv/Fm, over the
Figure 1. (A):time
incubation Kinetics
underof the microalgal
different light photosynthetic efficiency,
intensities (0–400 −2 s−1 ) in in
µmol mexpressed anFv/Fm, over the incu-
air atmosphere with
bation
0.04% CO time2 under
(p < different
0.05 for each light
day).intensities
(B): (0–400
Microalgal μmol
biomass m −2 s −1 )
concentrationin an air atmosphere
expressed in µL with 0.04%
PCV/mL
CO (p <sixth
on 2the 0.05 incubation
for each day).day,(B): Microalgal
over differentbiomass concentration
light intensities (0–400 expressed
µmol m−in2 μL s−1PCV/mL on the
). Significantly
sixth incubation day, over different light intensities (0–400 μmol m −2 s−1). Significantly different are
different are the biomass concentrations of the following treatments: 0 µmol m s versus 50, − 2 − 1
the biomass concentrations of the following 1treatments: 0 μmol −m2 −2 −s−1 versus 50, 100 and 400 μmol
100 and 400 µmol −2 m−−12 s−1 ; 10 µmol m−2−2s−−1 versus 50 µmol−2m −1 s 1 and 50 µmol m−2−2−1s−1 versus
m−2 s−1; 10 μmol m s versus 50 μmol m s and 50 μmol m s versus 200 μmol m s (p < 0.05).
200 µmol m−2 s−1 (p < 0.05).

The effect of light on the molecular structure and function of the photosynthetic ap-
paratus of the microalga Chlorella vulgaris is presented in more detail in Figure 2, which
depicts a series of more specific OJIP parameters. The results reveal that stress was induced
by increasing the light intensity under ambient CO2 concentration in closed cultivation sys-
tems. Concisely, the density of active photosynthetic reaction centers (RC/CS0 ) decreased
 The effect of light on the molecular structure and function of the photosynthetic ap-
paratus of the microalga Chlorella vulgaris is presented in more detail in Figure 2, which
Symmetry 2021, 13, 997 6 of 16
depicts a series of more specific OJIP parameters. The results reveal that stress was in-
duced by increasing the light intensity under ambient CO2 concentration in closed culti-
vation systems. Concisely, the density of active photosynthetic reaction centers (RC/CS0)
decreased
while while size
the antenna the antenna
per activesize per active
reaction centerreaction
(ABS/RC) center (ABS/RC)
increased, increased,
leading leading
to decreased
to decreased
primary primary photochemistry
photochemistry (PSI0)dissipation
(PSI0 ) and increased and increased dissipation
energy per activeenergy percenter
reaction active
reaction
(DI0 /RC). center
As a (DI
result,0 /RC).
the As a result,
performance the
indexperformance
(PI (abs) ) index
decreased (PI
(Figure
(abs)) decreased
2). The (Figure
parameters 2).
The parameters
mentioned above arementioned
the mostabove are the most
representative representative
indicators indicators of the sensitiv-
of the sensitivity/tolerance of the
photosynthetic
ity/tolerance of apparatus under abiotic
the photosynthetic stress and
apparatus are inabiotic
under agreement
stresswithandtheareobservations
in agreement
made
with intheabiotic stress conditions,
observations such as
made in abiotic ozone
stress elevation such
conditions, [52], as
high
ozoneUVBelevation
radiation[52],
[53,54]
high
and
UVB low temperature
radiation [53,54] [55].
and low temperature [55].

Figure 2. Changes in RC/CS0 , ABS/RC, PSI0 , DI0 /RC and PI(abs) (OJIP test) over the 6 days of incubation time compared
toFigure 2. Changes invalues
the corresponding RC/CSof 0, ABS/RC, PSI0, different
day 0, under DI0/RC and PI(abs)
light (OJIP test)
intensities over µmol
(0–400 m−2 sof−1incubation
the 6 days ) in an air time compared
atmosphere to the
(0.04%
COcorresponding
2 ). (A): 0 µmolvalues
m −2ofs−
day 0,
1 , (B):under
10 different
µmol m −2 light
s −1 , intensities
(C): 50 (0–400
µmol m −μmol
2 s−1 ,m(D):
−2 s−1) in
100 an air
µmol atmosphere
m (0.04%
−2 s−1 , (E): 200 CO2). (A):
µmol m −20 μmol
s−1
m −2 s−1, (B): 10 μmol m−2 s−1, (C): 50 μmol m−2 s−1, (D): 100 μmol m−2 s−1, (E): 200 μmol m−2 s−1 and (F): 400 μmol m−2 s−1.
and (F): 400 µmol m s . − 2 − 1

3.2.Effect
3.2. EffectofofVarious
Various Extreme
Extreme CO CO 2 Concentrations
2 Concentrations at at
thethe Photosynthetic
Photosynthetic Mechanism
Mechanism under
under Several
Light Intensities
Several Light Intensities
AAseries
seriesofofvarious
variousextreme
extremeCO CO2 concentrations
2 concentrations(0.04,
(0.04,10,
10,20,
20,30,
30,4040andand60%)
60%)under
under
different
different light
lightintensities
intensities(0,(0,
10,10,
50,50,
100, 200200
100, and 400400
and µmol
μmolm−m s−s1−1) )were
2 −2
werecombined
combinedininorder
order
tototest
testthe
theeffects of of
effects elevated
elevatedCOCO2 concentrations in the
2 concentrations Chlorella
in the vulgaris
Chlorella cultures
vulgaris (Figure
cultures 3).
(Figure
The results showed that under continuous darkness (0 µmol m −2 s−2−1 ), by increasing the
3). The results showed that under continuous darkness (0 μmol m s ), by increasing the −1

CO2 levels, the photosynthetic efficiency (Fv/Fm) decreased proportionally. However, in

the presence of light, changes induced in the Fv/Fm were completely different. Rising CO2
levels (up to 40%; 1000 times higher than the atmospheric concentration) had a protective
role to the photosynthetic apparatus, expressed with higher Fv/Fm values (Figure 3), in
contrast to the light-induced stress effect that appeared in treatments without any additional
 CO2 levels, the photosynthetic efficiency (Fv/Fm) decreased proportionally. However,
the presence of light, changes induced in the Fv/Fm were completely different. Rising C
Symmetry 2021, 13, 997 7 of 16
levels (up to 40%; 1000 times higher than the atmospheric concentration) had a protecti
role to the photosynthetic apparatus, expressed with higher Fv/Fm values (Figure 3),
contrast to the light-induced stress effect that appeared in treatments without any add
CO2 (Figure tional CO2 above-mentioned
3). The (Figure 3). The above-mentioned
protective mechanism protective mechanism
induced by elevatedinduced
COby 2 elevat
CO2 allowed
allowed microalgae microalgae
to grow even attohighgrow even
light at high light
intensities µmol m−2(400
(400intensities s−1 ),μmol m−2 s−1a), inducin
inducing
a newsymmetry.
new functional functionalThat symmetry. That was under
was impossible impossible
ambientunderCOambient CO2 concentration.
2 concentration. CO2 C
concentrations at 60% andathigher
concentrations 60% and werehigher werefor
not ideal notthe
ideal for the microalgae,
microalgae, since they accounte
since they accounted
for an additional
for an additional stress factor stress
in eachfactor in each
tested lighttested lightexcept
intensity, intensity, except
the 100 µmol the −2 s−
m100 1.
μmol m−2 s
This light
This light intensity intensity
seems to be seems
the only toone
be the only for
suitable onemicroalgal
suitable for microalgal
adaptation, evenadaptation,
at 60% even
CO2 , since60%
the Fv/Fm
CO2, sincevaluesthewere
Fv/Fm maintained
values were to the levels of 40%
maintained COlevels
to the 2 and were
of 40% higher
CO2 and we
than the corresponding
higher than the values in the treatment
corresponding valueswith ambient
in the CO2with
treatment concentration.
ambient CO Lower
2 concentratio
intensitiesLower
(<100 µmol m−2 s(<100
intensities −1 ) were not enough
μmol m−2 s−1) were for not
efficient
enoughusefor
of the extremely
efficient use ofhigh
the extreme
CO2 concentration −2 s−1 ), in combination
high CO2(60%), while higher
concentration (60%),intensities
while higher (>100 µmol m(>100
intensities μmol m−2 s−1), in combinatio
with the excess
with of
theCO 2 , caused
excess of COfurther
2, causedstress effects.
further stress effects.

Figure 3. KineticsFigure 3. Kinetics photosynthetic

of the microalgal of the microalgal photosynthetic
efficiency, efficiency,
expressed expressed
in Fv/Fm, over theinincubation
Fv/Fm, over the
time incuba-
under different
light intensities tion time
(0–400 μmolunder
m−2 s−1different
) in an air light intensities
atmosphere with(0–400
different
µmolCOm2−concentrations
2 s−1 ) in an air (0.04–60%)
atmosphere(pwith different
< 0.05 for each light
intensity and eachCOday).
2 concentrations (0.04–60%) (p < 0.05 for each light intensity and each day).

For the analytical

For the parameters of the fluorescence
analytical parameters induction measurements
of the fluorescence (OJIP-test)(OJIP-tes
induction measurements
presented presented
in Figure 4,inthree different
Figure 4, threestates werestates
different chosenwerein order
chosen toin
study
ordertheto combined
study the combine
effect of light and
effect CO2 and
of light concentration in closedincultivation
CO2 concentration of Chlorella
systemssystems
closed cultivation vulgaris
of Chlorella vulgaris cu
cultures: (a) the ambient
tures: CO2 state
(a) the ambient COat 0.04%
2 state atCO 2 ; (b)
0.04% COthe concentration
2; (b) of 30% of
the concentration CO30%2 , with
CO2, with t
the most protective effect against
most protective high light
effect against highintensities (Figure
light intensities 3); and
(Figure 3);(c)
andthe(c)extremely
the extremely hig
high concentration of 60% CO2 , where the CO2 stress on the photosynthetic apparatus was
detected. In the absence of light, obvious signs of stress were observed in the presence
of high CO2 concentrations. In contrast, there was an improvement of the functionality
of the photosynthetic apparatus under light conditions combined with high CO2 levels
(30%). Briefly, the density of the reaction centers (RC/CS0 ) increased and the size of the
Symmetry 2021, 13, 997 8 of 16

concentration of 60% CO2, where the CO2 stress on the photosynthetic apparatus was de-
Symmetry 2021, 13, 997 tected. In the absence of light, obvious signs of stress were observed in the presence 8 ofof
16
high CO2 concentrations. In contrast, there was an improvement of the functionality of the
photosynthetic apparatus under light conditions combined with high CO2 levels (30%).
Briefly, the density of the reaction centers (RC/CS0) increased and the size of the antenna
antenna
per activeper activecenter
reaction reaction center (ABS/RC)
(ABS/RC) decreased
decreased in in the treatments
the treatments with 30% COwith 30% CO
2 compared2

to the corresponding treatments with ambient (0.04%) and extremely high (60%) CO2 (60%)
compared to the corresponding treatments with ambient (0.04%) and extremely high con-
CO2 concentrations.
centrations. Theseimproved
These changes changes the
improved
primarythephotochemistry
primary photochemistry (PSI0 ) and
(PSI0) and decreased
decreased
the the non-photochemical
non-photochemical energy dissipation
energy dissipation per active
per active reaction reaction
center centerwhich
(DI0/RC), (DI0 /RC),
led
which led to an increased photosynthetic performance (PI
to an increased photosynthetic performance (PI(abs)) (Figure(abs)
4). ) (Figure 4).

Figure
Figure4.4.Changes
Changes in in
RC/CS 0, ABS/RC,
RC/CS PSI0,PSI
0 , ABS/RC, DI00/RC
, DIand PIand
0 /RC (abs) (OJIP
PI(abs)test) over
(OJIP theover
test) 6 daystheof6incuba-
days of
tion time compared
incubation to the corresponding
time compared values of
to the corresponding day 0,ofunder
values day 0,different light intensities
under different (0–400
light intensities
μmol m −2 s−1) in an air atmosphere with 0.04% CO2, 30% CO2 and 60% CO2.
− 2 − 1
(0–400 µmol m s ) in an air atmosphere with 0.04% CO , 30% CO and 60% CO .
2 2 2

Comparing and combining all the above-mentioned results, the high concentration
of 30% CO2 was perfectly managed by the microalgal photosynthetic mechanism up to
the light intensity of 100 µmol m−2 s−1 . Higher light intensities were not so beneficial
 Comparing and combining all the above-mentioned results, the high concentration
of 30% CO2 was perfectly managed by the microalgal photosynthetic mechanism up to
Symmetry 2021, 13, 997 9 of 16
the light intensity of 100 μmol m−2 s−1. Higher light intensities were not so beneficial for the
microalga, since the non-photochemical energy dissipation was increased, mainly in the
first incubation day. After that incubation time interval, there was some adaptation signs,
which
for thecould permitsince
microalga, the microalgal survival in high
the non-photochemical energylight intensitieswas
dissipation (upincreased,
to 400 μmol m−2
mainly
sin),the
−1 andfirst
high CO2 concentrations
incubation day. After(up thattoincubation
60%) (Figure time 4).interval,
It is worth mentioning
there was somethat the
adapta-
stress effect that
tion signs, which appeared in the photosynthetic
could permit the microalgal apparatus survival ininhigh 60% light
CO2 was quenched
intensities (up in to
the
400light m−2 s−1 of
µmolintensity 100 high
), and μmolCO m2−2concentrations
s−1, compared (up to lowerto 60%) and higher4).light
(Figure intensities,
It is worth men-
meaning
tioning that that the
the stress
green effect
microalga Chlorella vulgaris
that appeared could also survive
in the photosynthetic in extremely
apparatus in 60%high CO2
CO concentrations, changing its bioenergetic strategy.− 2
was quenched in the light intensity of 100 µmol m s , compared to lower and higher
2
− 1

light intensities, meaning that the green microalga Chlorella vulgaris could also survive in
3.3. Photosynthetic
extremely high CO and Respiratory Activities
2 concentrations, changing under itsDifferent
bioenergetic Lightstrategy.
Intensities and CO2
Concentrations
3.3. Photosynthetic and Respiratory Activities under Different Light Intensities and CO2 Concentrations
The photosynthetic and respiratory activities of the microalga Chlorella vulgaris,
grownThe in closed systems under
photosynthetic a combination
and respiratory of six
activities different
of the lightChlorella
microalga intensities and six
vulgaris, dif-
grown
ferent CO concentrations, were calculated (Figure 5). It was
in closed systems under a combination of six different light intensities and six different
2 proven that the photosyn-
thetic rate, expressed were
CO2 concentrations, as μLcalculated
O2 (μL PCV) −1 h−1, was almost six times higher at extreme CO2
(Figure 5). It was proven that the photosynthetic rate,
levels
expressed(fromas20% µL toO260%)
(µL PCV) −
compared1 −
h to1 the almost
, was ambientsix levels
times(0.04%)
higher(Figure
at extreme 5). InCO the case
2 levels
that
(from the20%
photosynthetic
to 60%) compared activitytowastheexpressed
ambient levels per chlorophyll
(0.04%) (Figure content,
5). the corresponding
In the case that the
values at extreme
photosynthetic CO2 levels
activity also increased
was expressed almost eightcontent,
per chlorophyll times with the same trendvalues
the corresponding (data
not shown). Furthermore,
at extreme CO2 levels also taking into consideration
increased almost eightthe times overall
withresults
the samefor each
trend individual
(data not
light
shown).intensity, it was clearly
Furthermore, takingshown that the maximal
into consideration photosynthetic
the overall rate was
results for each reached
individual at
light
intensity,
light it wasofclearly
intensities about shown
100–200thatμmol themmaximal
−2 s . Higher
−1 photosynthetic
light intensitiesrate was reached
induced at light
photoinhi-
intensities of about −2 s−1 . Higher light intensities induced photoinhibition
bition (Figure 4) and100–200
decreasedµmol themphotosynthetic activity (Figure 5). It is remarkable that
(Figure CO
extreme 4) and decreased the
2 concentrations photosynthetic
strongly induced theactivity (Figure 5).
photosynthetic It is (Figure
activity remarkable that
5) at low
extreme
light CO2 concentrations
intensities of 10–50 μmolstrongly induced
m−2 s−1, while the the photosynthetic
measurements wereactivity (Figure
definitely better 5)atat
lowhigh
the light intensities
light intensities of 10–50
(100–200 µmolμmol m−m s−s1−1, ),while
2 −2 the measurements
highlighting the extremely werehigh definitely
photo-
better at the high light intensities (100–200 µmol m −2 s−1 ), highlighting the extremely high
synthetic sensitivity/efficiency of this microalga and its increased demands for CO2 con-
photosynthetic
sumption. sensitivity/efficiency
Chlorella vulgaris is supposed of this
to bemicroalga and its increased
a very adaptable demandsthat
green microalga for sur-
CO2
vives in adverseChlorella
consumption. vulgaris
conditions, is supposed
changing to be a very
its bioenergetic adaptable
strategy, green microalga
depending on the best that
survives in adverse
combination conditions, changing
of CO2 concentration and lightitsintensity.
bioenergetic strategy, depending on the best
combination of CO2 concentration and light intensity.

Photosynthetic andrespiratory
respiratoryactivities
activitiesof
ofmicroalgae
microalgae expressed
expressed in −1 −1
Figure
Figure 5. 5.
Photosynthetic and μL OΟ₂
in µL μLPCV
2 µL PCV−1 h
h−1
cultivatedinindifferent
differentlight
lightintensities
intensities (0–400
(0–400μmol
µmolmm −2 s−1 ) in an air atmosphere with different CO
cultivated −2 s−1) in an air atmosphere with different CO2 con-2
concentrations
centrations (0.04–60%)
(0.04–60%) (p < for
(p < 0.05 0.05each
for each
light light intensity,
intensity, each each
CO2 CO 2 concentration
concentration and each
and each day).day).

Kinetics of total O2 quantities per culture during the entire incubation time are pre-
sented in Figure 6. The trend of the measurements confirmed the above-mentioned photo-
synthetic activity results. Even though significant amounts of O2 were produced at very low
light intensities (10 µmol m−2 s−1 ) over the incubation period of six days, the maximum
O2 production was reached at a light intensity of about 100–200 µmol m−2 s−1 . Increasing
Symmetry 2021, 13, 997 10 of 16

Kinetics of total O2 quantities per culture during the entire incubation time are pre-
Symmetry 2021, 13, 997 sented in Figure 6. The trend of the measurements confirmed the above-mentioned10pho- of 16
tosynthetic activity results. Even though significant amounts of O2 were produced at very
low light intensities (10 μmol m−2 s−1) over the incubation period of six days, the maximum
O2 production was reached at a light intensity of about 100–200 μmol m−2 s−1. Increasing
CO2 concentrations improved photosynthetic
photosynthetic O O22 production, even at extreme
extreme COCO22 values.
Treatments
Treatmentswith
with30–40%
30–40%COCO22 yielded
yielded the
the optimal
optimal results
results in
in our
our closed
closed cultivation
cultivation systems
systems
(Figure
(Figure 6). Notably, in higher light intensities, 20% CO22 was consumed and converted to
6). Notably, in higher light intensities, 20% CO was consumed and converted to
O in almost
O22 in almost 2–3
2–3 days,
days, while
while by
by the
the end
end of
of the
the 4–6
4–6 days
days ofof incubation,
incubation, concentrations
concentrations as as
high
high as
as 40%
40% CO were almost
CO22 were almost completely
completely converted
converted toto O
O22..

Figure 6. Kinetics of total O2 production per microalgal culture over the incubation time under dif-
Figure 6. Kinetics of total O2 production −2 sper
−1 ) microalgal culture over the incubation time under dif-
ferent light intensities (0–400 µmol m in an air atmosphere with different CO2 concentrations
ferent light intensities (0–400 μmol m −2 s−1) in an air atmosphere with different CO2 concentrations (0.04–
(0.04–60%). (A): 0 µmol m−2 s−1 , (B): 10 µmol m−2 s−1 , (C): 50 µmol m−2 s−1 , −2 (D):
−1, 100 µmol m−2 s−2−1−1,
60%). (A): 0 μmol−m2 −2 s−−11, (B): 10 μmol m−2 s−1, (C): 50 μmol m −2 s−1, (D): 100 μmol m s (E): 200 μmol m s
(E):
and200
(F):µmol m ms−2 s−1and
400 μmol (p < (F):
0.05400
for µmol m−2 intensity
each light s−1 (p < 0.05
andfor each
each light intensity and each day).
day).
3.4. Microalgal Biomass Production under Different Light Intensities and CO2 Concentrations
3.4. Microalgal Biomass Production under Different Light Intensities and CO2 Concentrations
The microalgal biomass concentration under several light intensities and CO2 concen-
Theismicroalgal
trations presented in biomass
Figure 7. concentration undershow
The results clearly severalthatlight intensities
the optimal and CO
biomass 2 con-
increase
centrations is presented in Figure 7. The results clearly show that the optimal
(about four times higher than the initial cell biomass) was recorded at Chlorella vulgaris cul- biomass
increase
tures, (about
which werefour times higher
incubated in closedthan the initial
cultivation cell biomass)
systems, was recorded
when exposed to lightat Chlorella
intensities
vulgaristhan
higher cultures, which
50 µmol m were
− 2 − 1
s and incubated in closed cultivation
CO2 concentrations of about systems,
30–40%when
CO2 . exposed
Increasingto
lightlight
the intensities higher
intensity to 100than
µmol 50m μmol
−2 s−m1 ,−2200
s−1 and
µmolCO m2−concentrations
2 s−1 and 400 µmolof about
m−230–40%
s−1 ledCOto a2.
slight improvement of biomass productivity, whereas increasing the CO2 to 60% CO2 lim-
ited the microalgal biomass production (Figure 7). This extreme CO2 concentration (60%)
changed the bioenergetic management of Chlorella vulgaris cultures. The microalga limited
the energy yields for growth (Figure 7), in order to face the extra stress (Figures 3 and 4).
Oppositely, 30–40% CO2 concentrations were ideal for the closed cultivation systems used
 Increasing the light intensity to 100 μmol m−2 s−1, 200 μmol m−2 s−1 and 400 μmol m−2 s−1 led
to a slight improvement of biomass productivity, whereas increasing the CO2 to 60% CO2
limited the microalgal biomass production (Figure 7). This extreme CO2 concentration
Symmetry 2021, 13, 997 (60%) changed the bioenergetic management of Chlorella vulgaris cultures. The microalga 11 of 16
limited the energy yields for growth (Figure 7), in order to face the extra stress (Figures 3
and 4). Oppositely, 30–40% CO2 concentrations were ideal for the closed cultivation sys-
tems used
in this in this contribution,
contribution, leading
leading to the to the best
best growth growth
(Figure (Figure
7) and 7) and photochemistry
photochemistry (Figures 3–6),
(Figures 3–6), mainly to high light intensities (100–200
− 2 −μmol
1 m −2 s−1). Lower CO2 concen-
mainly to high light intensities (100–200 µmol m s ). Lower CO2 concentrations did
trations
not seem didtonot seemChlorella
satisfy to satisfyvulgaris
Chlorella vulgaris demands,
demands, leading toleading
a limitedto functionality
a limited function-
of the
ality of the photosynthetic apparatus
photosynthetic apparatus and limited growth.and limited growth.

Microalgalbiomass
Figure7.7.Microalgal
Figure biomassconcentration
concentrationexpressed
expressedininμL PCV/mLononthe
µLPCV/mL thesixth
sixthincubation
incubationday,
day,
over different light intensities (0–400 µmol m −2 s−1 ) and CO concentrations (0.04–60%) (p < 0.05 for
over different light intensities (0–400 μmol m s ) and CO2 concentrations
−2 −1 2 (0.04–60%) (p < 0.05 for
eachlight
each lightintensity,
intensity,each
eachCO
CO2 2concentration
concentrationand
andeach
eachday).
day).

Toevaluate
To evaluatethe thequality
qualityofofthe
thebiomass
biomassproduced,
produced,we weexamined
examinedthe thelevels
levelsofofcarbohy-
carbohy-
drates and lipids. The microalgae content in carbohydrates and lipids
drates and lipids. The microalgae content in carbohydrates and lipids was measured was measured in the
in
three different “carbon states” mentioned above
the three different “carbon states” mentioned above (0.04% CO(0.04% CO –30% CO
2 2–30% CO –60% CO
2 2–60% CO ) under
2 2) un-
different
der light
different conditions
light (Tables
conditions 1 and
(Tables 2). In
1 and 2).the
In absence of light,
the absence microalgae
of light, did not
microalgae didhave
not
any exploitable
have external
any exploitable energy
external source,
energy thus thus
source, they they
supplied their their
supplied demand by breaking
demand down
by breaking
their cellular stocks in carbohydrates and lipids, and therefore lower values were recorded.
down their cellular stocks in carbohydrates and lipids, and therefore lower values were
The same tendency was observed in all CO2 -deprived cultures at all tested light intensities.
recorded. The same tendency was observed in all CO2-deprived cultures at all tested light
On the contrary, optimizing the CO2 concentration at 30%, led to an accumulation of
intensities. On the contrary, optimizing the CO2 concentration at 30%, led to an accumu-
carbohydrates at approximately 30–37% of dry weight in the first incubation day (Table 1).
lation of carbohydrates at approximately 30–37% of dry weight in the first incubation day
No significant changes in the cellular lipid level between the treatments were observed,
(Table 1). No significant changes in the cellular lipid level between the treatments were
even under high light conditions (Table 2). Interestingly, pushing CO2 levels up to 60% led
observed, even under high light conditions (Table 2). Interestingly, pushing CO2 levels up
to an accumulation of lipids increase of up to 19% of dry weight in the first incubation day
to 60% led to an accumulation of lipids increase of up to 19% of dry weight in the first
under high light intensities (Table 2). This observation could be attributed to the microalgal
incubation day under high light intensities (Table 2). This observation could be attributed
attempt to face the excess stress caused by the extremely high CO2 concentration of 60%.
to the microalgal attempt to face the excess stress caused by the extremely high CO2 con-
Biomass increase was restricted, due to the lipid accumulation for saving energy in a more
centration of 60%. Biomass increase was restricted, due to the lipid accumulation for sav-
valuable form.
ing energy in a more valuable form.
Symmetry 2021, 13, 997 12 of 16

Table 1. Percentage (% dry weight) of total carbohydrate content over 0, 1, 3 and 6 days of incubation under 0.04% CO2 ,
30% CO2 and 60% CO2 under different light intensities (0–400 µmol m−2 s−1 ).

Carbohydrates (% DW) ± SE% Carbohydrates (mg/L) ± SE%

[CO2 ] Light Intensity
Day 0 Day 1 Day 3 Day 6 Day 0 Day 1 Day 3 Day 6
0 µmol m−2 s−1 24.0 ± 1.0 17.9 ± 0.5 15.2 ± 1.2 13.5 ± 0.7 42.2 ± 1.8 31.5 ± 0.9 26.7 ± 2.0 23.7 ± 1.1
10 µmol m−2 s−1 24.0 ± 1.0 17.9 ±1.0 8.6 ± 0.4 10.2 ± 0.2 42.2 ± 1.8 31.5 ± 1.8 23.1 ± 0.7 30.5 ± 0.2
0.04%

50 µmol m−2 s−1 24.0 ± 1.0 14.6 ± 0.9 11.9 ± 1.0 11.3 ± 1.1 42.2 ± 1.8 26.1 ± 1.5 28.8 ± 2.5 30.5 ± 3.0
100 µmol m−2 s−1 24.0 ± 1.0 17.1 ± 1.2 12.5 ± 2.2 12.1 ± 1.0 42.2 ± 1.8 30.0 ± 2.2 26.4 ± 4.6 27.7 ± 2.2
200 µmol m−2 s−1 24.0 ± 1.0 20.1 ± 2.2 18.5 ± 0.4 18.4 ± 1.8 42.2 ± 1.8 35.3 ± 3.9 35.9 ± 1.0 38.8 ± 3.7
400 µmol m−2 s−1 24.0 ± 1.0 11.9 ± 0.4 8.6 ± 0.1 10.0 ± 0.1 42.2 ± 1.8 20.9 ± 0.8 16.8 ± 0.3 21.0 ± 0.4
0 µmol m−2 s−1 24.0 ± 1.0 21.5 ± 2.9 21.4 ± 1.2 15.0 ± 0.5 42.2 ± 1.8 37.8 ± 5.1 37.6 ± 3.4 26.4 ± 1.0
10 µmol m−2 s−1 24.0 ± 1.0 17.1 ± 2.7 35.5 ± 0.7 23.4 ± 0.7 42.2 ± 1.8 70.8 ± 4.3 79.6 ± 1.7 73.1 ± 2.0
50 µmol m−2 s−1
30%

24.0 ± 1.0 37.2 ± 1.3 14.0 ± 1.6 18.7 ± 1.0 42.2 ± 1.8 51.8± 3.3 90.2 ± 6.9 116.2 ± 5.8
100 µmol m−2 s−1 24.0 ± 1.0 31.0 ± 1.7 20.1 ± 2.0 15.5 ± 3.0 42.2 ± 1.8 79.0 ± 4.2 114.3 ± 8.5 104.1±20.3
200 µmol m−2 s−1 24.0 ± 1.0 34.8 ± 1.1 19.4 ± 0.6 21.4 ± 0.5 42.2 ± 1.8 67.1 ± 2.7 83.4 ± 1.9 138.6 ± 3.2
400 µmol m−2 s−1 24.0 ± 1.0 34.4 ± 2.3 28.7 ± 2.8 16.5 ± 0.7 42.2 ± 1.8 87.8 ± 5.7 151.7±14.6 112.9± 4.7
0 µmol m−2 s−1 24.0 ± 1.0 19.5 ± 0.7 19.0 ± 0.5 15.6 ± 0.4 42.2 ± 1.8 34.3 ± 1.2 32.9 ± 1.0 27.4 ± 0.8
10 µmol m−2 s−1 24.0 ± 1.0 14.3 ± 0.4 14.8 ± 0.7 12.2 ± 0.8 42.2 ± 1.8 27.6 ± 0.9 56.4 ± 1.5 33.7 ± 2.3
50 µmol m−2 s−1
60%

24.0 ± 1.0 27.0 ± 0.4 21.5 ± 2.0 22.8 ± 2.6 42.2 ± 1.8 52.3 ± 0.7 60.6 ± 5.8 85.4 ± 9.7
100 µmol m−2 s−1 24.0 ± 1.0 27.1 ± 2.0 20.7 ± 1.3 16.1 ± 1.6 42.2 ± 1.8 52.4 ± 4.0 68.1 ± 4.2 74.4 ± 7.4
200 µmol m−2 s−1 24.0 ± 1.0 26.3 ± 1.0 22.8 ± 1.5 29.1 ± 1.2 42.2 ± 1.8 67.5 ± 1.8 72.2 ± 4.7 111.3 ± 4.8
400 µmol m−2 s−1 24.0 ± 1.0 29.6 ± 1.8 27.3 ± 0.7 16.1 ± 0.4 42.2 ± 1.8 62.7 ± 3.7 73.5 ± 2.1 78.0 ± 1.2

Table 2. Percentage (% dry weight) of total lipid content over 0, 1, 3 and 6 days of incubation under 0.04% CO2 , 30% CO2
and 60% CO2 under different light intensities (0–400 µmol m−2 s−1 ).

Lipids (% DW) ± SE% Lipids (mg/L) ± SE%

[CO2 ] Light Intensity
Day 0 Day 1 Day 3 Day 6 Day 0 Day 1 Day 3 Day 6
0 µmol m−2 s−1 13.2 ± 0.6 8.90 ± 0.4 13.1 ± 0.2 11.1 ± 0.1 23.2 ± 0.8 15.6 ± 0.7 23.0 ± 0.4 19.5 ± 0.1
10 µmol m−2 s−1 13.2 ± 0.6 14.2 ± 0.2 9.60 ± 1.0 9.70 ± 0.2 23.2 ± 0.8 25.1 ± 0.3 16.9 ± 1.8 19.2 ± 0.4
0.04%

50 µmol m−2 s−1 13.2 ± 0.6 12.4 ± 0.5 10.3 ± 0.2 8.50 ± 0.3 23.2 ± 0.8 20.3 ± 1.2 21.8 ± 0.7 25.1 ± 1.3
100 µmol m−2 s−1 13.2 ± 0.6 13.5 ± 0.6 13.5 ± 0.7 10.6 ± 0.7 23.2 ± 0.8 23.7 ± 1.0 28.5 ± 1.5 24.2 ± 1.6
200 µmol m−2 s−1 13.2 ± 0.6 13.3 ± 0.6 11.0 ± 0.1 9.30 ± 0.1 23.2 ± 0.8 23.4 ± 1.0 21.3 ± 0.3 19.7 ± 0.1
400 µmol m−2 s−1 13.2 ± 0.6 13.7 ± 1.0 10.5 ± 0.4 8.20 ± 0.7 23.2 ± 0.8 24.1 ± 1.7 20.4 ± 0.8 17.2 ± 1.5
0 µmol m−2 s−1 13.2 ± 0.6 12.0 ± 0.4 10.7 ± 0.4 12.4 ± 0.3 23.2 ± 0.8 21.0 ± 0.6 18.8 ± 0.7 21.8 ± 0.6
10 µmol m−2 s−1 13.2 ± 0.6 14.2 ± 0.4 9.30 ± 0.2 10.3 ± 0.2 23.2 ± 0.8 24.5 ± 0.8 20.9 ± 0.3 32.0 ± 0.6
50 µmol m−2 s−1
30%

13.2 ± 0.6 11.7 ± 0.4 8.30 ± 0.1 7.60 ± 0.4 23.2 ± 0.8 28.8 ± 1.5 35.1 ± 0.2 49.1 ± 0.9
100 µmol m−2 s−1 13.2 ± 0.6 10.4 ± 0.6 13.9 ± 0.8 8.60 ± 0.1 23.2 ± 0.8 26.6 ± 1.6 57.9 ± 3.2 58.2 ± 0.5
200 µmol m−2 s−1 13.2 ± 0.6 10.6 ± 0.7 7.20 ± 0.3 8.00 ± 0.2 23.2 ± 0.8 27.0 ± 1.7 36.2 ± 1.8 51.8 ± 1.3
400 µmol m−2 s−1 13.2 ± 0.6 13.6 ± 0.6 7.50 ± 0.3 6.80 ± 0.4 23.2 ± 0.8 34.8 ± 1.6 39.4 ± 1.8 46.2 ± 3.0
0 µmol m−2 s−1 13.2 ± 0.6 11.4 ± 0.5 11.2 ± 1.4 13.9 ± 0.8 23.2 ± 0.8 20.1 ± 0.8 19.8 ± 2.4 24.5 ± 1.4
10 µmol m−2 s−1 13.2 ± 0.6 15.8 ± 0.1 12.3 ± 0.4 14.0 ± 0.1 23.2 ± 0.8 30.7 ± 0.2 31.0 ± 1.2 38.6 ± 0.2
50 µmol m−2 s−1
60%

13.2 ± 0.6 13.8 ± 0.2 11.2 ± 0.1 11.7 ± 0.4 23.2 ± 0.8 26.7 ± 0.4 31.5 ± 0.5 44.0 ± 2.2
100 µmol m−2 s−1 13.2 ± 0.6 16.4 ±0.5 18.3 ± 0.4 8.60 ± 0.2 23.2 ± 0.8 31.8 ± 0.9 62.0 ± 1.3 39.8 ± 1.0
200 µmol m−2 s−1 13.2 ± 0.6 19.0 ± 0.5 10.5 ± 0.3 8.80 ± 0.3 23.2 ± 0.8 24.6 ± 1.0 33.2 ± 0.9 33.7 ± 1.1
400 µmol m−2 s−1 13.2 ± 0.6 13.4 ± 1.1 13.6 ± 0.4 8.10 ± 0.3 23.2 ± 0.8 28.4 ± 2.3 36.7 ± 1.0 24.2 ± 0.8

4. Discussion
The present study examined the metabolic and functional differentiation of the green
alga Chlorella vulgaris under various light intensities and extreme CO2 concentrations. The
results verified the ability of this species to modify hostile CO2 atmospheres by converting
CO2 to O2 , through the photosynthetic management of solar radiation, for the benefit of
the organism, producing a microalgal biomass rich in carbohydrates and lipids.
In the current study, we found that high CO2 concentrations, even at extreme levels
up to 40%, not only increased biomass yield of Chlorella vulgaris, but also protected the
photosynthetic mechanism against photoinhibition and photodamage. Consequently, the
microalga Chlorella vulgaris is a species that does not only tolerate extremely high CO2
levels, but also, extremely high CO2 levels (up to 1500 times higher concentration than
the ambient atmospheric CO2 concentration) strongly increase its photosynthetic activity.
Symmetry 2021, 13, 997 13 of 16

The result is a pronounced CO2 conversion into O2 and valuable microalgal biomass. The
potentiality of this photosynthetic system for CO2 mitigation showed clearly that today’s
Earth atmospheric CO2 levels are not the optimal habitat for Chlorella vulgaris.
In this work, we report optimal growth at extreme CO2 concentrations (30–40%) with
inhibitory effects appearing first at 60% CO2 , where a completely different bioenergetic
strategy was used by the microalga in order to face the extra stress effect caused by the
extreme CO2 concentration. In a hermitically sealed cultivation system similar to ours,
Papazi et al. [23] found that Chlorella minutissima grew best at around 25% CO2 , verifying
the CO2 tolerance of Chlorella sp. [23]. However, Chlorella minutissima was evidently
stressed at 40% CO2 , while Chlorella vulgaris did not show any stress effects at that level [23].
Since Chlorella vulgaris demands extremely high CO2 concentrations in order to yield the
best biomass production and mitigates up to 288 L CO2 L PCV−1 day−1 , it is ideal for
direct biological CO2 capture from industrial fuel gases, such as in coal plants that usually
generate 10–20% CO2 fumes, and other similar applications [56].
In our experiments, in light intensities above 50 µmol m−2 s−1 , CO2 concentrations up
to 30% and 40% were almost completely converted to O2 in 2–3 and 4–6 days, respectively.
Additionally, the microalgal culture quadrupled its biomass in just 5–6 days without any
additional energy input. The microalgal biomass consists of relatively high amounts of
carbohydrates and lipids, ideal for biofuel production, without significant differences in
the cellular concentrations (20–37% carbohydrates and 13–19% lipids), considering light
intensity and CO2 concentration (Tables 1 and 2). However, extreme CO2 concentrations
led to a strong increase in carbohydrate (about 3.5 times higher amount in 3 days) and lipid
(about 2.5 times higher amount in 3 days) accumulation per culture (Tables 1 and 2).
Large scale cultivation of Chlorella vulgaris could bring valuable biomass yield, while
also solving environmental problems, mainly CO2 -driven climate change. The aforemen-
tioned is in agreement with recent techno-economic analyses and life-cycle assessments of
microalgae-based production systems, suggesting the necessity of using the microalgae
biomass in an integrated biorefinery system from which all the valuable components are
extracted and utilized [57].
It is known that the annual global CO2 emissions since 2000 consist of 34.07 × 109 t CO2
from fossil fuel and industry emissions, and 5.86 × 109 t CO2 from land-use change emis-
sions. As a result, the total CO2 emissions are 39.93 × 109 t/year or 0.11 × 109 t/day [58].
The importance of the present study lies in the fact that the microalga Chlorella vulgaris is
not only resistant to extremely high CO2 concentrations, but actually requires them for
intense photosynthetic activity without any stress induction. These data highlight that
this particular microalga is a unique tool for solving global warming. If we consider its
excellent photosynthetic activity (12 L O2 L PCV−1 h−1 or 288 L O2 L PCV−1 day−1 ) when
exposed to 20 or 30% CO2 , and the ratio of 1 L CO2 gas = 0.001812 kg CO2 , a theoretical
huge Chlorella vulgaris culture of 210 Km3 (210 × 109 m3 ) consisting of a cell density of 1 µL
PCV/mL culture could mitigate the total global CO2 emissions, converting them to O2 and
valuable biomass.
In recent decades, space missions have seen revolutionary progress, and sending
humans to other planets, such as Mars, is now a realistic and most anticipated goal. NASA
is already equipped with, and constantly updates, Environmental Control and Life Sup-
port Systems (ECLSS) for water recovery, air revitalization and oxygen generation [59].
In addition to that, bioregenerative life-support systems (BLSS) are developed with the
aim of continuously recycling the above resources, while generating food via oxygenic
photosynthetic microorganisms [60]. Most importantly, these systems aim for constant O2
regeneration from CO2 -rich atmospheres [61], in which the results of the present contri-
bution may seem helpful. Chlorella vulgaris is a photosynthetic microalga that demands
extreme CO2 concentrations for its metabolism, having the ability to convert a CO2 -rich,
hostile-for-humans atmosphere to a hospitable O2 -rich atmosphere. As illustrated in
Figure 5, the maximal rate of O2 production happened in the treatments with CO2 concen-
trations of 20% and more, under a light intensity of 100–200 µmol m−2 s−1 , and reached a
Symmetry 2021, 13, 997 14 of 16

value of about 288 L O2 L PCV−1 day−1 . These results could be extrapolated and compared
with a regular human’s oxygen requirements (about 590 L O2 day−1 ) [62], which could
be completely covered from a Chlorella vulgaris culture consisting from approximately 2 L
PCV microalgae. The continuous recycling of a human settlement’s atmosphere in another
planet, such as Mars, in which O2 is consumed and CO2 is produced continuously, could
be provided by a closed cultivation system of Chlorella vulgaris, creating an efficient and
sustainable O2 regeneration system for cosmonauts and even settlers on other planets.

Author Contributions: Conceptualization, K.K.; supervision, K.K.; investigation, F.M., A.P. and K.K.;
funding acquisition, K.K.; methodology, F.M., A.P. and K.K.; resources, K.K.; validation, F.M. and
A.P.; visualization, F.M. and A.P.; writing—original draft, F.M.; writing—review and editing, K.K.
and A.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research has been co-financed by the European Regional Development Fund of the
European Union and Greek national funds through the Operational Program Competitiveness,
Entrepreneurship and Innovation, under the call Special Actions in aquatic farming—industrial
materials—open innovation in culture. Project code: T6ΥBΠ-00494 (MIS 5056221).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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