The Microalga Chlorella Vulgaris As a Natural Bioenergetic System for Effective CO2 Mitigation—New Perspectives Against Global Warming

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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 *

Department of Biology, University of Crete, Voutes University Campus, 70013 Heraklion, Greece; [email protected] (F.M.); [email protected] (A.P.) * Correspondence: [email protected]; 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 −2 −1 light intensities (0–400 µmol m s ) 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 −1 −1 288 L CO2 L PCV day under optimal conditions, therefore, this microalga can be used for direct Citation: Mountourakis, F.; Papazi, biological CO -reducing strategies and other green biotechnological applications. All of the above A.; Kotzabasis, K. The Microalga 2 Chlorella vulgaris Chlorella vulgaris as a Natural suggest that is one of the most prominent competitors for a closed algae-powered bioreactor that is able to consume huge amounts of CO . Thus, it is a sustainable and natural Bioenergetic System for Effective CO2 2 Mitigation—New Perspectives bioenergetic system with perspectives in dealing with major environmental issues such as global against Global Warming. Symmetry warming. In addition, Chlorella vulgaris cultures could also be used as bioregeneration systems in 2021, 13, 997. https://doi.org/ extraterrestrial missions for continuous atmospheric recycling of the human settlements, paving the 10.3390/sym13060997 way for astrobiological applications.

Academic Editor: Keywords: Chlorella vulgaris; CO2 mitigation; global warming; biofuels; photosynthesis; environmen- Eleftherios Touloupakis tal biotechnology; astrobiology

Received: 9 May 2021 Accepted: 1 June 2021 Published: 2 June 2021 1. Introduction

Publisher’s Note: MDPI stays neutral Over the last couple of centuries, industrialization along with urbanization have with regard to jurisdictional claims in brought an immense increase in greenhouse gases (GHGs), mostly carbon dioxide (CO2), published maps and institutional afﬁl- which is considered one of the main causes of global warming [1] and other major environ- iations. 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 CO every year, as opposed to almost 10 megatons 2 centuries ago [6]. There is already Copyright: © 2021 by the authors. 2 Licensee MDPI, Basel, Switzerland. clear evidence that anthropogenic emissions of GHGs, such as CO2, alter the natural carbon This article is an open access article cycle, which leads to an accelerated warming of our planet [7]. Based on Representative distributed under the terms and Concentration Pathways (RCPs), adopted by the International Panel on Climate Change conditions of the Creative Commons (IPCC), and other modelling scenarios, scientists predict that the amount of CO2 in the Attribution (CC BY) license (https:// atmosphere might double or even triple at the end of the 21st century [8,9]. This will result ◦ creativecommons.org/licenses/by/ in a planet that is hotter by more than 4 C compared to preindustrial ages [8,9], well above ◦ 4.0/). the 2 C danger mark agreed in COP21 Paris Agreement [10].

Symmetry 2021, 13, 997. https://doi.org/10.3390/sym13060997 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 9 H2O as a reducing agent and releases O2, evolved 3.8 × 10 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 unicellular 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 production 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 corresponding 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 −1 −1 respiratory activity was expressed as µLO2 µL PCV h . 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 chloroform: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 H2SO4 (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 H3PO4 85%, 125 mL ddH2O 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 ddH2O. At 400 µL of the diluted samples, we added 900 µL of thymol- sulfuric acid reagent (0.1 g thymol into 100 mL of H2SO4 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. Symmetry 2021, 13, 997 ANOVA One Way and Tukey HSD paired tests were used for testing the significance of5 of the 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 apparatusΤhe effects of Chlorella of various vulgaris lightunder intensities natural (0–400 atmospheric μmol m−2 conditionss−1) on the photosynthetic in closed cultivation appa- ratussystems of Chlorella were evaluated. vulgaris under The kineticsnatural atmospheric of the maximal conditions photosynthetic in closed efficiencycultivation (Fv/Fm) systems werein Figure evaluated.1A showed The kinetics that increased of the maximal light intensity photosynthetic acts as efficiency a major stress(Fv/Fm) factor in Figure for the 1A showedphotosynthetic that increased mechanism light intens underity ambient acts as a COmajor2 (0.04%) stress factor concentration. for the photosynthetic In complete mech- dark- anismness (0 underµmol ambient m−2 s− CO1), Fv/Fm2 (0.04%) remained concentration. constantly In complete high. darkness After a couple(0 μmol of m days,−2 s−1), Fv/Fm under remainedextremely constantly low light high. conditions After a(10 coupleµmol of mdays,−2 s −under1), Fv/Fm extremely values low showed light conditions a slight de-(10 μcrease.mol m As−2 s− light1), Fv/Fm intensity values increased showed toa slight 50 µmol decrease. m−2 s −As1, light the photosynthetic intensity increased efficiency to 50 μ dropmol mcame−2 s−1, earlier the photosynthetic and sharper, efficiency leadingto drop a decrease came earlier in photosynthetic and sharper, leading capacity. to a Stepping decreaseup in photosyntheticto 100 µmol m −capacity.2 s−1 and Stepping 200 µmol up m to− 2100s− 1μ,mol Fv/Fm m−2 decreaseds−1 and 200 downμmol tom− 0.42 s−1 (Figure, Fv/Fm1 A).de- −2 −1 −2 −1 creasedSpecifically, down at to400 0.4 µ(Figuremol m 1A).s Specifically,under ambient at 400 μ COmol2, m there s wasunder a totalambient breakdown CO2, there of was the aphotosynthetic total breakdown apparatus of the photosynthetic after the fourth apparatus incubation after daythe fourth (no reliable incubation measurements day (no reliable with measurementsOJIP-test; no observed with OJIP-test; bullets no in observed Figure1A). bullets These in Figure first results 1A). These showed first an results extremely showed light an extremelysensitive photosyntheticlight sensitive photosynthetic mechanism under mechanism ambient under carbon ambient dioxide carbon with dioxide limited with biomass lim- itedincrease biomass (Figure increase1B). (Figure 1B).

Figure 1. (A): Kinetics of the microalgal photosynthetic efﬁciency, expressed in Fv/Fm, over the Figureincubation 1. (A): timeKinetics under of the different microalgal light photosynthetic intensities (0–400 efficiency,µmol m expressed−2 s−1) in in an Fv/Fm, air atmosphere over the incu- with −2 −1 bation0.04% COtime2 (underp < 0.05 different for each light day). intensities (B): Microalgal (0–400 biomass μmol m concentration s ) in an air expressed atmosphere in µ withL PCV/mL 0.04% COon2 the (p < sixth 0.05 incubationfor each day). day, (B over): Microalgal different biomass light intensities concentration (0–400 expressedµmol m −in2 μs−L1 PCV/mL). Signiﬁcantly on the −2 −1 sixthdifferent incubation are the day, biomass over concentrationsdifferent light intensities of the following (0–400 μ treatments:mol m s ). 0 Significantlyµmol m−2 s− different1 versus are 50, the biomass concentrations of the following treatments: 0 μmol m−2 s−1 versus 50, 100 and 400 μmol 100 and 400 µmol m−2 s−1; 10 µmol m−2 s−1 versus 50 µmol m−2 s−1 and 50 µmol m−2 s−1 versus m−2 s−1; 10 μmol m−2 s−1 versus 50 μmol m−2 s−1 and 50 μmol m−2 s−1 versus 200 μmol m−2 s−1 (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 apparatus of the microalga Chlorella vulgaris is presented in more detail in Figure2, 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 systems. Concisely, the density of active photosynthetic reaction centers (RC/CS0) decreased Symmetry 2021, 13, 997 6 of 16

The effect of light on the molecular structure and function of the photosynthetic apparatus 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 induced by increasing the light intensity under ambient CO2 concentration in closed cultivation systems. Concisely, the density of active photosynthetic reaction centers (RC/CS0) whiledecreased the antenna while sizethe antenna per active size reaction per active center re (ABS/RC)action center increased, (ABS/RC) leading increased, to decreased leading primaryto decreased photochemistry primary photochemistry (PSI0) and increased (PSI0) dissipation and increased energy dissipation per active energy reaction per center active (DIreaction0/RC). center As a result, (DI0/RC). the performance As a result, indexthe performance (PI(abs)) decreased index (PI (Figure(abs)) decreased2). The parameters (Figure 2). mentionedThe parameters above arementioned the most above representative are the most indicators representative of the sensitivity/tolerance indicators of the sensitiv- of the photosyntheticity/tolerance of apparatus the photosynthetic under abiotic apparatu stress ands under are in abiotic agreement stress with and theare observationsin agreement madewith inthe abiotic observations stress conditions, made in abiotic such asstress ozone conditions, elevation such [52], as high ozone UVB elevation radiation [52], [53, 54high] andUVB low radiation temperature [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 the corresponding 2. Changes in values RC/CS of0, ABS/RC, day 0, under PSI0, differentDI0/RC and light PI(abs) intensities (OJIP test) (0–400 over µthemol 6 days m 2 sof 1incubation) in an air time atmosphere compared (0.04% to the corresponding values−2 of −day1 0, under different−2 light−1 intensities (0–400− μ2mol−1 m−2 s−1) in an air atmosphere−2 −1 (0.04% CO2). (A):− 20 μ−mol1 CO2). (A): 0 µmol m s ,(B): 10 µmol m s ,(C): 50 µmol m s ,(D): 100 µmol m s ,(E): 200 µmol m s −2 −1 −2 −1 −2 −1 −2 −1 −2 −1 −2 −1 andm ( Fs):, 400(B): µ10mol μmol m− m2 s− s1., (C): 50 μmol m s , (D): 100 μmol m s , (E): 200 μmol m s and (F): 400 μmol m s .

3.2.3.2. Effect Effect of of Various Various Extreme Extreme CO CO2 Concentrations2 Concentrations at at the the Photosynthetic Photosynthetic Mechanism Mechanism under under Several LightSeveral Intensities Light Intensities

AA series series of of various various extreme extreme CO CO2 concentrations2 concentrations (0.04, (0.04, 10, 10, 20, 20, 30, 30, 40 40 and and 60%) 60%) under under − − differentdifferent light light intensities intensities (0, (0, 10, 10, 50, 50, 100, 100, 200 200 and and 400 400µmol μmol m m2 −s2 s1−)1) were were combined combined in in order order toto test test the the effects effects of of elevated elevated CO CO2 concentrations2 concentrations in thein theChlorella Chlorella vulgaris vulgariscultures cultures (Figure (Figure3). −2 −1 The3). resultsThe results showed showed that that under un continuousder continuous darkness darkness (0 µ mol(0 μmol m ms−2 s),−1), by by increasing increasing the the 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 (Figure3), in contrast to the light-induced stress effect that appeared in treatments without any additional Symmetry 2021, 13, 997 7 of 16

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 Symmetry 2021, 13, 997 7 of 16 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 addi- CO2 (Figuretional3). The CO2 above-mentioned (Figure 3). The above-mentioned protective mechanism protective induced mechanism by elevated induced CO by2 elevated −2 −1 −2 −1 allowed microalgaeCO2 allowed to grow microalgae even at to high grow light even intensities at high light (400 intensitiesµmol m (400s ),μmol inducing m s a), inducing new functionala new symmetry. functional Thatsymmetry. was impossible That was underimpossible ambient under CO ambient2 concentration. CO2 concentration. CO2 CO2 concentrationsconcent at 60%rations and at higher 60% and were higher not ideal were for not the ideal microalgae, for the microalgae, since they accountedsince they accounted for an additionalfor an additional stress factor stress in each factor tested in each light tested intensity, light except intensity, the 100exceptµmol the m 100−2 s −μ1mol. m−2 s−1. This light intensityThis light seems intensity to be seems the only to one be the suitable only for one microalgal suitable for adaptation, microalgal even adaptation, at 60% even at CO2, since60% the Fv/FmCO2, since values the were Fv/Fm maintained values were to the maintained levels of 40% to the CO levels2 and wereof 40% higher CO2 and were than the correspondinghigher than the values corresponding in the treatment values with in the ambient treatment CO 2withconcentration. ambient CO Lower2 concentration. − − intensitiesLower (<100 µintensitiesmol m 2 s(<1001) wereμmol not m enough−2 s−1) were for not efﬁcient enough use for of theefficient extremely use of high the extremely −2 −1 CO2 concentrationhigh CO2 (60%),concentration while higher (60%), intensities while higher (>100 intensitiesµmol m (>100s μ),mol in combinationm−2 s−1), in combination with the excesswith ofthe CO excess2, caused of CO further2, caused stress further effects. stress effects.

Figure 3. KineticsFigure of the microalgal 3. Kinetics photosynthet of the microalgalic efficiency, photosynthetic expressed efﬁciency, in Fv/Fm, expressed over the in incubation Fv/Fm, over time the under incuba- different −2 −1 light intensities tion(0–400 timeμmol under m−2 s− different1) in an air lightatmosphere intensities with (0–400 differentµmol CO m2 concentrationss ) in an air (0.04–60%) atmosphere (p with < 0.05 different for each light intensity and eachCO day).2 concentrations (0.04–60%) (p < 0.05 for each light intensity and each day).

For the analyticalFor the parametersanalytical parameters of the ﬂuorescence of the fluorescence induction measurementsinduction measurements (OJIP-test) (OJIP-test) presentedpresented in Figure4 ,in three Figure different 4, three states different were states chosen were in order chosen to in study order the to combined study the combined effect of lighteffect and of light CO2 andconcentration CO2 concentration in closed in cultivation closed cultivation systems systems of Chlorella of Chlorella vulgaris vulgaris cul- cultures: (a)tures: the ambient(a) the ambient CO2 state CO at2 state 0.04% at CO0.04%2; (b) CO the2; (b) concentration the concentration of 30% of CO 30%2, with CO2, with the the most protectivemost protective effect againsteffect against high light high intensitieslight intensities (Figure (Figure3); and 3); (c) and the (c) extremely the extremely high 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 presence8 of of 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 perantenna active perreaction active center reaction (ABS/RC) center (ABS/RC)decreased in decreased the treatments in the treatmentswith 30% CO with2 compared 30% CO2 tocompared the corresponding to the corresponding treatments with treatments ambient with (0.04%) ambient and (0.04%) extremely and high extremely (60%)high CO2 (60%)con- CO concentrations. These changes improved the primary photochemistry (PSI ) and centrations.2 These changes improved the primary photochemistry (PSI0) and decreased0 decreased the non-photochemical energy dissipation per active reaction center (DI /RC), the non-photochemical energy dissipation per active reaction center (DI0/RC), which0 led which led to an increased photosynthetic performance (PI ) (Figure4). to an increased photosynthetic performance (PI(abs)) (Figure(abs) 4).

FigureFigure 4. 4. ChangesChanges in inRC/CS RC/CS0, ABS/RC,0, ABS/RC, PSI0, PSIDI00/RC, DI and0/RC PI and(abs) (OJIP PI(abs) test)(OJIP over test) the over6 days the of 6 incuba- days of tionincubation time compared time compared to the corresponding to the corresponding values of values day 0, of under day 0, different under different light intensities light intensities (0–400 μmol m−2 s−1) in −an2 air−1 atmosphere with 0.04% CO2, 30% CO2 and 60% CO2. (0–400 µmol m s ) in an air atmosphere with 0.04% CO2, 30% CO2 and 60% CO2.

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 Symmetry 2021, 13, 997 9 of 16

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, whichfor the could microalga, permit since the microalgal the non-photochemical survival in high energy light dissipation intensities was (up increased,to 400 μmol mainly m−2 sin−1), theand first high incubation CO2 concentrations day. After (up that to incubation 60%) (Figure time 4). interval, It is worth there mentioning was some that adapta- the stresstion signs,effect that which appeared could permit in the photosynthetic the microalgal apparatus survival in in high 60% lightCO2 was intensities quenched (up in to −2 −1 −2 −1 the400 lightµmol intensity m s of), and100 highμmol CO m2 concentrations s , compared (upto lower to 60%) and (Figure higher4). light It is worthintensities, men- meaningtioning thatthat thethe stressgreen effectmicroalga that appearedChlorella vulgaris in the photosynthetic could also survive apparatus in extremely in 60% high CO2 −2 −1 COwas2 concentrations, quenched in the changing light intensity its bioenergetic of 100 µmol strategy. m s , compared to lower and higher light intensities, meaning that the green microalga Chlorella vulgaris could also survive in 3.3.extremely Photosynthetic high CO and2 concentrations, Respiratory Activities changing under its Different bioenergetic Light strategy.Intensities and CO2 Concentrations 3.3. Photosynthetic and Respiratory Activities under Different Light Intensities and CO Concentrations The photosynthetic and respiratory activities of the microalga Chlorella2 vulgaris, grownThe in closed photosynthetic systems under and respiratory a combination activities of six of thedifferent microalga lightChlorella intensities vulgaris and six, grown dif- ferentin closed CO2 systemsconcentrations, under a were combination calculated of six(Figure different 5). It lightwas intensitiesproven that and the six photosyn- different −1 −1 theticCO2 concentrations,rate, expressed wereas μL calculated O2 (μL PCV) (Figure h 5, ).was It wasalmost proven six times that the higher photosynthetic at extreme CO rate,2 −1 −1 levelsexpressed (from as 20%µLO to2 60%)(µL PCV)comparedh to, was the almostambient six levels times (0.04%) higher (Figure at extreme 5). In CO the2 levels case that(from the 20% photosynthetic to 60%) compared activity to was the expressed ambient levels per chlorophyll (0.04%) (Figure content,5). Inthe the corresponding case that the valuesphotosynthetic at extreme activity CO2 levels was expressedalso increased per chlorophyllalmost eight content, times with the correspondingthe same trend values(data notat extremeshown). Furthermore, CO2 levels also taking increased into consider almostation eight the times overall with results the same for each trend individual (data not lightshown). intensity, Furthermore, it was clearly taking shown into consideration that the maximal the overall photosynthetic results for eachrate was individual reached light at lightintensity, intensities it was of clearly about shown100–200 that μmol the m maximal−2 s−1. Higher photosynthetic light intensities rate wasinduced reached photoinhi- at light bitionintensities (Figure of about4) and 100–200 decreasedµmol the m photosynthetic−2 s−1. Higher activity light intensities (Figure induced5). It is remarkable photoinhibition that extreme(Figure 4CO) and2 concentrations decreased the strongly photosynthetic induced the activity photosynthetic (Figure5). activity It is remarkable (Figure 5) at that low lightextreme intensities CO2 concentrations of 10–50 μmol stronglym−2 s−1, while induced the themeasurements photosynthetic were activity definitely (Figure better5) at − − thelow high light light intensities intensities of 10–50(100–200µmol μmol m m2 −s2 s1−1,), while highlighting the measurements the extremely were high definitely photo- −2 −1 syntheticbetter at thesensitivity/efficiency high light intensities of this (100–200 microalgaµmol mand itss increased), highlighting demands the extremely for CO2 con- high sumption.photosynthetic Chlorella sensitivity/efficiency vulgaris is supposed of thisto be microalga a very adap andtable its increased green microalga demands that for sur- CO2 vivesconsumption. in adverseChlorella conditions, vulgaris changingis supposed its bioenergetic to be a very strategy, adaptable depending green microalga on the best that combinationsurvives in adverseof CO2 concentration conditions, changing and light its intensity. bioenergetic strategy, depending on the best combination of CO2 concentration and light intensity.

−1 −1 FigureFigure 5. 5.PhotosyntheticPhotosynthetic and and respiratory respiratory activi activitiesties of of microalgae microalgae expressed in µμLOL Ο₂2 µμLL PCVPCV−1 h−1 −2 −1 cultivatedcultivated in in different different light light intensities intensities (0–400 (0–400 μµmolmol m m−2 s−1) ins an) air in anatmosphere air atmosphere with different with different CO2 con- CO2 centrationsconcentrations (0.04–60%) (0.04–60%) (p < 0.05 (p < for 0.05 each for eachlight lightintensity, intensity, each eachCO2 COconcentration2 concentration and each and eachday). day).

Kinetics of total O2 quantities per culture during the entire incubation time are presented in Figure6. The trend of the measurements conﬁrmed the above-mentioned photosynthetic 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 −2 −1 O2 production was reached at a light intensity of about 100–200 µmol m s . 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-mentioned10 pho- 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 OO22 production, even at extreme COCO22 values. Treatments withwith 30–40%30–40% COCO22 yielded the optimal results inin our closed cultivation systems (Figure(Figure6 6).). Notably,Notably, in in higher higher light light intensities, intensities, 20% 20% CO CO22 waswas consumedconsumed andand convertedconverted toto O2 in almost 2–3 days, while by the endend ofof thethe 4–64–6 daysdays ofof incubation,incubation, concentrationsconcentrations asas high as 40% CO2 werewere almost almost completely completely converted to O 22..

Figure 6. Kinetics of total O2 production per microalgal culture over the incubation time under dif- Figure 6. Kinetics of total O2 production−2 per−1 microalgal culture over the incubation time under different light intensities (0–400 µmol m s ) 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,(D): 100 µmol m−2 s−1, 60%). (A): 0 μmol 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 µ −2 −1 µ −2 −1 (andE): 200(F): 400mol μmol m ms−2 s−1 and(p < (0.05F): 400 for eachmol light m intensitys (p <0.05 and foreach each day). light intensity and each 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- trationsThe is microalgal presented inbiomass Figure 7concentration. The results clearly under show several that light the optimalintensities biomass and CO increase2 con- (aboutcentrations four timesis presented higher thanin Figure the initial 7. The cell re biomass)sults clearly was recordedshow that at theChlorella optimal vulgaris biomasscul- tures,increase which (about were four incubated times higher in closed than cultivation the initial systems, cell biomass) when exposed was recorded to light at intensities Chlorella vulgaris cultures, which− 2were−1 incubated in closed cultivation systems, when exposed to higher than 50 µmol m s and CO2 concentrations of about 30–40% CO2. Increasing thelight light intensities intensity higher to 100 thanµmol 50 mμmol−2 s −m1,−2 200 s−1 andµmol CO m2− concentrations2 s−1 and 400 µ ofmol about m− 230–40%s−1 led CO to a2. slight improvement of biomass productivity, whereas increasing the CO2 to 60% CO2 limited the microalgal biomass production (Figure7). This extreme CO 2 concentration (60%) changed the bioenergetic management of Chlorella vulgaris cultures. The microalga limited

the energy yields for growth (Figure7), in order to face the extra stress (Figures3 and4). Oppositely, 30–40% CO2 concentrations were ideal for the closed cultivation systems used Symmetry 2021, 13, 997 11 of 16

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 microalga11 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- temsin this used contribution, in this contribution, leading to theleading best growthto the best (Figure growth7) and (Figure photochemistry 7) and photochemistry ( Figures3–6 ), (Figures 3–6), mainly to high light intensities (100–200−2 − μ1mol m−2 s−1). Lower CO2 concen- mainly to high light intensities (100–200 µmol m s ). Lower CO2 concentrations did trationsnot seem did to not satisfy seemChlorella to satisfy vulgaris Chlorellademands, vulgaris demands, leading to leading a limited to functionalitya limited function- of the alityphotosynthetic of the photosynthetic apparatus apparatus and limited and growth. limited growth.

µ FigureFigure 7. 7. MicroalgalMicroalgal biomass biomass concentration concentration expressed expressed in in μLL PCV/mL PCV/mL on on the the sixth sixth incubation incubation day, day, µ −2 −1 overover different different light light intensities intensities (0–400 (0–400 μmol m−2 ss−1) and) and CO CO2 concentrations2 concentrations (0.04–60%) (0.04–60%) (p ( p<< 0.05 0.05 for for eacheach light light intensity, intensity, each each CO CO2 2concentrationconcentration and and each each day). day).

ToTo evaluate evaluate the the quality quality of of the the biomass biomass produced, produced, we we examined examined the the levels levels of of carbohy- carbohy- dratesdrates and and lipids. lipids. The The microalgae contentcontent inincarbohydrates carbohydrates and and lipids lipids was was measured measured in thein three different “carbon states” mentioned above (0.04% CO –30% CO –60% CO ) under the three different “carbon states” mentioned above (0.04% CO2 2–30% CO2 2–60% CO2 2) un- derdifferent different light light conditions conditions (Tables (Tables1 and 1 2and). In 2). the In absence the absence of light, of light, microalgae microalgae did not did have not haveany exploitableany exploitable external external energy energy source, source, thus thus they they supplied supplied their their demand demand by breaking by breaking down 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 (Table1). 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 (Table2). Interestingly, pushing CO 2 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 (Table2). 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 centrationvaluable form.of 60%. Biomass increase was restricted, due to the lipid accumulation for saving 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, −2 −1 30% CO2 and 60% CO2 under different light intensities (0–400 µmol m s ).

Carbohydrates (% DW) ± SE% Carbohydrates (mg/L) ± SE% [CO ] Light Intensity 2 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 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 −2 −1 ± ± ± ± ± ± ± ± 0.04% 100 µmol m s 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 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 − − 30% 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 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 − − 60% 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 −2 −1 and 60% CO2 under different light intensities (0–400 µmol m s ).

Lipids (% DW) ± SE% Lipids (mg/L) ± SE% [CO ] Light Intensity 2 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 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 −2 −1 ± ± ± ± ± ± ± ± 0.04% 100 µmol m s 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 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 − − 30% 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 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 − − 60% 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 −1 −1 best biomass production and mitigates up to 288 L CO2 L PCV day , 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]. −2 −1 In our experiments, in light intensities above 50 µmol m s , 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 (Tables1 and2). However, extreme CO 2 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 (Tables1 and2). 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]. 9 It is known that the annual global CO2 emissions since 2000 consist of 34.07 × 10 t CO2 9 from fossil fuel and industry emissions, and 5.86 × 10 t CO2 from land-use change emis- 9 9 sions. As a result, the total CO2 emissions are 39.93 × 10 t/year or 0.11 × 10 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 −1 −1 −1 −1 excellent photosynthetic activity (12 L O2 L PCV h or 288 L O2 L PCV day ) 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 contribution 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 Figure5 , the maximal rate of O2 production happened in the treatments with CO2 concentrations 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

−1 −1 value of about 288 L O2 L PCV day . These results could be extrapolated and compared −1 with a regular human’s oxygen requirements (about 590 L O2 day )[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.

References

1. Peter, S.C. Reduction of CO2 to Chemicals and Fuels: A Solution to Global Warming and Energy Crisis. ACS Energy Lett. 2018, 3, 1557–1561. [CrossRef] 2. McNeil, B.I.; Matear, R.J. Southern Ocean acidification: A tipping point at 450-ppm atmospheric CO2. Proc. Natl. Acad. Sci. USA 2008, 105, 18860–18864. [CrossRef] 3. Etheridge, D.M.; Steele, L.P.; Langenfelds, R.L.; Francey, R.J.; Barnola, J.-M.; Morgan, V.I. Natural and anthropogenic changes in atmospheric CO2 over the last 1000 years from air in Antarctic ice and firn. J. Geophys. Res. Atmos. 1996, 101, 4115–4128. [CrossRef] 4. Earth System Research Laboratory National Oceanic and Atmospheric Administration. Available online: https://www.esrl.noaa. gov/gmd/ccgg/trends/ (accessed on 4 May 2021). 5. Lüthi, D.; Le Floch, M.; Bereiter, B.; Blunier, T.; Barnola, J.-M.; Siegenthaler, U.; Raynaud, D.; Jouzel, J.; Fischer, H.; Kawamura, K.; et al. High-resolution carbon dioxide concentration record 650,000–800,000 years before present. Nature 2008, 453, 379–382. [CrossRef] 6. Ritchie, H.; Roser, M. CO2 and Greenhouse Gas Emissions. 2020. Available online: https://ourworldindata.org/co2-and-other- greenhouse-gas-emissions (accessed on 1 August 2020). 7. Szulejko, J.E.; Kumar, P.; Deep, A.; Kim, K.-H. Global warming projections to 2100 using simple CO2 greenhouse gas modeling and comments on CO2 climate sensitivity factor. Atmos. Pollut. Res. 2017, 8, 136–140. [CrossRef] 8. Nazarenko, L.; Schmidt, G.A.; Miller, R.L.; Tausnev, N.; Kelley, M.; Ruedy, R.; Russell, G.L.; Aleinov, I.; Bauer, S.; Bleck, R.; et al. Future climate change under RCP emission scenarios with GISS ModelE2. J. Adv. Model. Earth Syst. 2015, 7, 244–267. [CrossRef] 9. Riahi, K.; van Vuuren, D.P.; Kriegler, E.; Edmonds, J.; O’Neill, B.C.; Fujimori, S.; Bauer, N.; Calvin, K.; Dellink, R.; Fricko, O.; et al. The Shared Socioeconomic Pathways and their energy, land use, and greenhouse gas emissions implications: An overview. Glob. Environ. Chang. 2017, 42, 153–168. [CrossRef] 10. Walsh, B.; Ciais, P.; Janssens, I.A.; Peñuelas, J.; Riahi, K.; Rydzak, F.; van Vuuren, D.P.; Obersteiner, M. Pathways for balancing CO2 emissions and sinks. Nat. Commun. 2017, 8, 14856. [CrossRef][PubMed] 11. Rosa, G.M.; Moraes, L.; Cardias, B.B.; Souza, M.; Costa, J. Chemical absorption and CO2 biofixation via the cultivation of Spirulina in semicontinuous mode with nutrient recycle. Bioresour. Technol. 2015, 192, 321–327. [CrossRef][PubMed] 12. El Mekawy, A.; Hegab, H.M.; Mohanakrishna, G.; Elbaz, A.F.; Bulut, M.; Pant, D. Technological advances in CO2 conversion electro-biorefinery: A step toward commercialization. Bioresour. Technol. 2016, 215, 357–370. [CrossRef][PubMed] 13. Duarte, J.H.; Fanka, L.S.; Costa, J.A.V. Utilization of simulated flue gas containing CO2, SO2, NO and ash for Chlorella fusca cultivation. Bioresour. Technol. 2016, 214, 159–165. [CrossRef] 14. Wang, B.; Li, Y.; Wu, N.; Lan, C.Q. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 2008, 79, 707–718. [CrossRef] 15. Singh, J.; Dhar, D.W. Overview of Carbon Capture Technology: Microalgal Biorefinery Concept and State-of-the-Art. Front. Mar. Sci. 2019, 6. [CrossRef] 16. Johnson, M.P. Photosynthesis. Essays Biochem. 2016, 60, 255–273. [CrossRef] 17. Buick, R. When did oxygenic photosynthesis evolve? Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008, 363, 2731–2743. [CrossRef] Symmetry 2021, 13, 997 15 of 16

18. Lyons, T.W.; Reinhard, C.T.; Planavsky, N.J. The rise of oxygen in Earth’s early ocean and atmosphere. Nature 2014, 506, 307–315. [CrossRef] 19. Keller, D.P.; Lenton, A.; Littleton, E.W.; Oschlies, A.; Scott, V.; Vaughan, N.E. The Effects of Carbon Dioxide Removal on the Carbon Cycle. Curr. Clim. Chang. Rep. 2018, 4, 250–265. [CrossRef][PubMed] 20. Chen, C.-Y.; Yeh, K.-L.; Aisyah, R.; Lee, D.-J.; Chang, J.-S. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour. Technol. 2011, 102, 71–81. [CrossRef][PubMed] 21. Gouveia, L.; Oliveira, A.C. Microalgae as a raw material for biofuels production. J. Ind. Microbiol. Biotechnol. 2008, 36, 269–274. [CrossRef] 22. Papazi, A.; Korelidou, A.; Andronis, E.; Parasyri, A.; Stamatis, N.; Kotzabasis, K. Bioenergetic reprogramming plasticity under nitrogen depletion by the unicellular green alga Scenedesmus Obliq. Planta 2018, 247, 679–692. [CrossRef][PubMed] 23. Papazi, A.; Makridis, P.; Divanach, P.; Kotzabasis, K. Bioenergetic changes in the microalgal photosynthetic apparatus by extremely high CO2 concentrations induce an intense biomass production. Physiol. Plant. 2008, 132, 338–349. [CrossRef] 24. Zerveas, S.; Kydonakis, E.; Moutidis, P.; Maragkoudakis, A.; Kotzabasis, K. Microalgae strategy in anoxic atmospheres with various CO2 concentrations—Environmental and (astro)biotechnological perspectives. Environ. Exp. Bot. 2021, 187, 104474. [CrossRef] 25. Anjos, M.; Fernandes, B.D.; Vicente, A.A.; Teixeira, J.A.; Dragone, G. Optimization of CO2 bio-mitigation by Chlorella vulgaris. Bioresour. Technol. 2013, 139, 149–154. [CrossRef] 26. Lam, M.K.; Lee, K.T. Effect of carbon source towards the growth of Chlorella vulgaris for CO2 bio-mitigation and biodiesel production. Int. J. Greenh. Gas Control 2013, 14, 169–176. [CrossRef] 27. Zheng, Y.; Li, F.; Hao, L.; Shedayi, A.A.; Guo, L.; Ma, C.; Huang, B.; Xu, M. The optimal CO2 concentrations for the growth of three perennial grass species. BMC Plant Biol. 2018, 18, 27. [CrossRef] 28. Safi, C.; Zebib, B.; Merah, O.; Pontalier, P.-Y.; Vaca-Garcia, C. Morphology, composition, production, processing and applications of Chlorella vulgaris: A review. Renew. Sustain. Energy Rev. 2014, 35, 265–278. [CrossRef] 29. Ru, I.T.K.; Sung, Y.Y.; Jusoh, M.; Wahid, M.E.A.; Nagappan, T. Chlorella vulgaris: A perspective on its potential for combining high biomass with high value bioproducts. J. Appl. Phycol. 2020, 1, 2–11. [CrossRef] 30. Görs, M.; Schumann, R.; Hepperle, D.; Karsten, U. Quality analysis of commercial Chlorella products used as dietary supplement in human nutrition. J. Appl. Phycol. 2009, 22, 265–276. [CrossRef] 31. Kholif, A.E.; Morsy, T.A.; Matloup, O.H.; Anele, U.Y.; Mohamed, A.G.; El-Sayed, A.B. Dietary Chlorella vulgaris microalgae improves feed utilization, milk production and concentrations of conjugated linoleic acids in the milk of Damascus goats. J. Agric. Sci. 2016, 155, 508–518. [CrossRef] 32. Ahmad, M.T.; Shariff, M.; Yusoff, F.; Goh, Y.M.; Banerjee, S. Applications of microalga Chlorella vulgaris in aquaculture. Rev. Aquac. 2018, 12, 328–346. [CrossRef] 33. Schreiber, C.; Schiedung, H.; Harrison, L.; Briese, C.; Ackermann, B.; Kant, J.; Schrey, S.D.; Hofmann, D.; Singh, D.; Ebenhoh, O.; et al. Evaluating potential of green alga Chlorella vulgaris to accumulate phosphorus and to fertilize nutrient-poor soil substrates for crop plants. J. Appl. Phycol. 2018.[CrossRef] 34. Faheed, F.A.; Abd-El Fattah, Z. Effect of Chlorella vulgaris as bio-fertilizer on growth parameters and metabolic aspects of lettuce plant. J. Agri. Soc. Sci. 2008, 4, 165–169. 35. Singh, A.; Nigam, P.S.; Murphy, J.D. Renewable fuels from algae: An answer to debatable land based fuels. Bioresour. Technol. 2011, 102, 10–16. [CrossRef] 36. Kim, K.H.; Choi, I.S.; Kim, H.M.; Wi, S.G.; Bae, H.-J. Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation. Bioresour. Technol. 2014, 153, 47–54. [CrossRef] 37. Asadi, P.; Rad, H.A.; Qaderi, F. Lipid and biodiesel production by cultivation isolated strain Chlorella sorokiniana pa.91 and Chlorella vulgaris in dairy wastewater treatment plant effluents. J. Environ. Health Sci. Eng. 2020, 18, 573–585. [CrossRef][PubMed] 38. Beyerinck, M.W. Culturversuche mit Zoochlorellen, Lichenengonidien und anderen niederen Algen. Bot. Ztg. 1890, 47, 725–739, 741–754, 757–768, 781–785. 39. Wong, Y.K.; Ho, Y.H.; Ho, K.C.; Leung, H.M.; Yung, K.K.L. Growth Medium Screening for Chlorella vulgaris Growth and Lipid Production. J. Aquac. Mar. Biol. 2017, 6, 00143. [CrossRef] 40. Nichols, H.W.; Bold, H.C. Trichosarcina polymorpha Gen. et Sp. Nov. J. Phycol. 1965, 1, 34–38. [CrossRef] 41. Serra-Maia, R.; Bernard, O.; Gonçalves, A.; Bensalem, S.; Lopes, F. Influence of temperature on Chlorella vulgaris growth and mortality rates in a photobioreactor. Algal Res. 2016, 18, 352–359. [CrossRef] 42. Seyfabadi, J.; Ramezanpour, Z.; Amini Khoeyi, Z. Protein, fatty acid, and pigment content of Chlorella vulgaris under different light regimes. J. Appl. Phycol. 2010, 23, 721–726. [CrossRef] 43. Zerveas, S.; Mente, M.; Tsakiri, D.; Kotzabasis, K. Microalgal photosynthesis induces alkalization of aquatic environment as + a result of H uptake independently from CO2 concentration—New perspectives for environmental applications. J. Environ. Manage. 2021, 289, 112546. [CrossRef] 44. Navakoudis, E.; Vrentzou, K.; Kotzabasis, K. A polyamine- and LHCII protease activity-based mechanism regulates the plasticity and adaptation status of the photosynthetic apparatus. Biochim. Biophys. Acta Bioenerg. 2007, 1767, 261–271. [CrossRef] Symmetry 2021, 13, 997 16 of 16

45. Strasser, B.J.; Strasser, R.J. Measuring Fast Fluorescence Transients to Address Environmental Questions: The JIP-Test. In Pho- tosynthesis: From Light to Biosphere; Mathis, P., Ed.; Kluwer Academic Press: Dordrecht, The Netherlands, 1995; pp. 977–980. [CrossRef] 46. Srivastava, A.; Strasser, R.J.; Govindjee. Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii. Photosynth. Res. 1995, 43, 131–141. [CrossRef] 47. Strasser, R.J.; Tsimilli-Michael, M.; Srivastava, A. Analysis of the Chlorophyll a Fluorescence Transient. In Chlorophyll a Fluorescence. Advances in Photosynthesis and Respiration; Papageorgiou, G.C., Govindjee, Eds.; Springer: Dordrecht, The Netherlands, 2004; Volume 19. [CrossRef] 48. Folch, J.; Lees, M.; Stanley, G.H.S. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 1957, 226, 497–509. [CrossRef] 49. Sati, H.; Mitra, M.; Mishra, S.; Baredar, P. Microalgal lipid extraction strategies for biodiesel production: A review. Algal Res. 2019, 38, 101413. [CrossRef] 50. Park, J.; Jeong, H. Easy and rapid quantification of lipid contents of marine dinoflagellates using the sulpho-phospho-vanillin method. Algae 2016, 31, 391–401. [CrossRef] 51. Schulze, C.; Wetzel, M.; Reinhardt, J.; Schmidt, M.; Felten, L.; Mundt, S. Screening of microalgae for primary metabolites including β-glucans and the influence of nitrate starvation and irradiance on β-glucan production. J. Appl. Phycol. 2016, 28, 2719–2725. [CrossRef] 52. Navakoudis, E.; Lütz, C.; Langebartels, C.; Lütz-Meindl, U.; Kotzabasis, K. Ozone impact on the photosynthetic apparatus and the protective role of polyamines. Biochim. Biophys. Acta. 2003, 1621, 160–169. [CrossRef] 53. Lütz, C.; Navakoudis, E.; Seidlitz, H.K.; Kotzabasis, K. Simulated solar irradiation with enhanced UV-B adjust plastid- and thylakoid-associated polyamine changes for UV-B protection. Biochim. Biophys. Acta 2005, 1710, 24–33. [CrossRef][PubMed] 54. Sfichi, L.; Ioanidis, E.N.; Kotzabasis, K. Thylakoid-associated polyamines adjust the UVB-sensitivity of the photosynthetic apparatus by means of LHCII changes. Photochem. Photobiol. 2004, 80, 499–506. [CrossRef] 55. Sfakianaki, M.; Sfichi, L.; Kotzabasis, K. The involvement of LHCII-associated polyamines in the response of the photosynthetic apparatus to low temperature. J. Photochem. Photobiol. B 2006, 84, 181–188. [CrossRef] 56. Jin, H.F.; Lim, B.R.; Lee, K. Influence of nitrate feeding on carbon dioxide fixation by microalgae. J. Environ. Sci. Health A 2006, 41, 2813–2824. [CrossRef][PubMed] 57. Chew, K.W.; Yap, J.Y.; Show, P.L.; Suan, N.H.; Juan, J.C.; Ling, T.C.; Lee, D.-J.; Chang, J.-S. Microalgae biorefinery: High value products perspectives. Bioresour. Technol. 2017, 229, 53–62. [CrossRef][PubMed] 58. Friedlingstein, P.; O’Sullivan, M.; Jones, M.W.; Andrew, R.M.; Hauck, J.; Olsen, A.; Peters, G.P.; Peters, W.; Pongratz, J.; Sitch, S.; et al. Global Carbon Budget. Earth Syst. Sci. Data 2020, 12, 3269–3340. [CrossRef] 59. Carrasquillo, R. ISS ECLSS Technology Evolution for Exploration. In Proceedings of the 43rd AIAA Aerospace Sciences Meeting and Exhibit, Reno, NV, USA, 10–13 January 2005. [CrossRef] 60. Yang, L.; Li, H.; Liu, T.; Zhong, Y.; Ji, C.; Lu, Q.; Fan, L.; Li, J.; Leng, L.; Li, K.; et al. Microalgae biotechnology as an attempt for bioregenerative life support systems: Problems and prospects. J. Chem. Technol. Biotechnol. 2019, 94, 3039–3048. [CrossRef] 61. Battistuzzi, M.; Cocola, L.; Salasnich, B.; Erculiani, M.S.; Alei, E.; Morosinotto, T.; Claudi, R.; Poletto, L.; La Rocca, N. A new remote sensing-based system for the monitoring and analysis of growth and gas exchange rates of photosynthetic microorganisms under simulated non-terrestrial conditions. Front. Plant Sci. 2020, 11, 182. [CrossRef][PubMed] 62. Jones, H. Design Rules for Space Life Support Systems. SAE Tech. Pap. 2003, 2356. [CrossRef]