Effects of CO2 enrichment on primary photochemistry, growth and astaxanthin accumulation in the chlorophyte Haematococcus pluvialis

Accepted Manuscript Effects of CO2 enrichment on primary photochemistry, growth and astaxanthin accumulation in the chlorophyte Haematococcus pluvialis

K. Chekanov, E. Schastnaya, A. Solovchenko, E. Lobakova PII: DOI: Reference:

S1011-1344(17)30336-6 doi: 10.1016/j.jphotobiol.2017.04.028 JPB 10807

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

10 March 2017 21 April 2017 24 April 2017

Please cite this article as: K. Chekanov, E. Schastnaya, A. Solovchenko, E. Lobakova , Effects of CO2 enrichment on primary photochemistry, growth and astaxanthin accumulation in the chlorophyte Haematococcus pluvialis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jpb(2017), doi: 10.1016/j.jphotobiol.2017.04.028

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Effects of CO2 enrichment on primary photochemistry, growth and astaxanthin accumulation in the chlorophyte Haematococcus pluvialis K. Chekanov a,b,*, E. Schastnaya a, A. Solovchenko a, E. Lobakova a a

Department of Bioengineering, Faculty of Biology, Moscow State University,

Moscow, Russia b

National Research Nuclear University MEPhi, Centre for Humanities Research and

Correspondence: Konstantin Chekanov, Department of Bioengineering, Faculty

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

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Technology, Moscow, Russia

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of Biology, Moscow State University,119234, GSP-1 Moscow, Russia. E-mail: [email protected]

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Abstract

The atmospheric CO2 level is limiting for growth of phototrophic organisms such as microalgae, so CO2 enrichment boosts the growth and photosynthesis of microalgal

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cultures. Still, excessive CO2 injection might inhibit photosynthesis of microalgae. We investigated the effect of continuous sparging of the cultures of Haematococcus pluvialis BM 1 (IPPAS H-2018) (Chlorophyceae), the richest natural source of the

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value-added pigment astaxanthin. H. pluvialis cultures with CO2-enriched air-gas

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mixtures (with CO2 level from the atmospheric to 20%) on growth and astaxanthin accumulation in the microalga. Special attention was paid to photosynthetic activity and non-photochemical excited chlorophyll states quenching in the microalgal cells, which

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was monitored via chlorophyll fluorescence analysis. We also report on the capability of CO2 capture by H. pluvialis derived from direct measurements of its elemental carbon

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content. The beneficial effect of the moderately high (5%) CO2 levels on the culture growth and astaxanthin accumulation under stress results in a higher overall astaxanthin productivity. However, increase of the CO2 level to 10% or 20% was deteriorative for growth, photosynthesis and carbon assimilation. The results support the possibility of combining a traditional two-stage H. pluvialis cultivation with CO2 bio-capture although a dilution of the flue gas before its injection is required.

Key words: Photosynthesis, Chlorophyll fluorescence, OJIP-curve, NPQ, Carbon assimilation.

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Abbreviations: Ψ0 – the probability of the electron transport beyond QA, AGM – air-gas mixture, AR – average removal, Car – carotenoids, CF – chlorophyll fluorescence, Chl – chlorophyll, DW – dry weight, ETC – electron transport chain, Fm, Fm' – maximal CF intensity of the dark-adapted and light-adapted state respectively, Fo – minimal CF intensity in dark adapted state, Fv – variable CF, φ(PSII)0 – maximum PSII photochemical quantum yield in dark adapted state, HL – light with high photon flux

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density (high light), Mo – the initial rise of OJIP-curve (the first derivative of V at t=0 μs), N(QA) – primary quinone acceptor turnover number, NPQ – the Stern-Volmer non-

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photochemical quenching parameter, PAR – photosynthetic active radiation, PFD – the photon flux density, PSA – photosynthetic apparatus, PSII – photosystem II, QA – PSII

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primary quinone electron acceptor, R – the volumetric removal (of CO2), Sm, Ss – normalized area above the curve in the case of multiple and single turnover number

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respectively, V – normalized fariable CF, VJ – relative height of OJ-step, ΔpH – the transmembrane proton gradient, Ψ0 – the probability of electron transport beyond QA–,

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ω(i) – the mass fraction of the element i.

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1. Introduction Contemporary increase in the atmospheric CO2 concentration, one of the most abundant greenhouse gases, is believed to give rise to the global warming effect [1]. Although vast amounts of CO2 are absorbed and released during natural processes in the biosphere, the recent dramatic net increase of CO2 is thought to be a consequence of the

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anthropogenic CO2 emission and a rapid deforestation [1,2]. Oxygenic phototrophic microorganisms (commonly termed as microalgae) are considered as a promising tool

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for capturing the technogenic CO2 and binding it in organic compounds via

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photosynthesis [3]. Moreover, since the atmospheric CO2 level is normally limiting for photosynthesis [4–6], CO2 enrichment of microalgal cultures boosts their productivity

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[7].

In the microalgal cell, CO2 is assimilated in the reaction catalyzed by ribulose1,5-bisphosphate carboxylase in the Calvin-Benson cycle [6,8,9]. Energy (in the form of

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ATP) and reducing equivalents (in the form of NADPH) for inorganic carbon assimilation are generated as a result of light reactions of photosynthesis, which start

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from primary photochemical processes (from the absorption of photosynthetic active radiation (PAR) quantum to charge separation and donation of the electron to the

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photosynthetic electron transport chain, ETC). On one hand, the parameters of primary photochemistry, efficiency of CO2-biocapture and algal productivity are expected to be interrelated, at list in the absence of factors limiting algal growth. On the other hand, the

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parameters characterizing primary photochemistry reflect closely the onset of and acclimation to stresses [8–13].

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Chlorophyll fluorescence (CF) signal e.g the transient curve of CF (OJIP) carries a lot of valuable information about primary photochemical processes in phototrophic organisms and on their physiological condition in general [8–13]. Accordingly, the CF analysis provides for estimation of the potential efficiency of carbon assimilation under different conditions, it is also very sensitive to the effects of adverse conditions [8–13]. The approach of express monitoring may be used for prediction of the stress effects on algal before appearance of other symptoms like irreversible damage to the photosynthetic apparatus [8–10,14,15]. In particular, the CF-derived parameters are useful for monitoring and optimization of the cultivation of microalgae [15–18].

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The microalga Haematococcus pluvialis (Chlorophyceae) is the richest source of natural astaxanthin [19], a ketocarotenoid very much sought after in the market as the most powerful natural antioxidant, a potent anti-inflammatory, anti-tumor, and neuroprotective agent. Dietary uptake of astaxanthin also determines the red coloration and hence the customer acceptance of salmon meat and crustacean shells, so it is extensively used as a quality feed of functional food additive, and as an ingredient in

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pharmaceutical formulations [19]. Biotechnological production of Astaxanthin from H. pluvialis involves the two-phase cultivation [19,20]. At the first phase (termed as the

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‘vegetative’ or ‘green’ stage), the cultivation conditions are conductive for vegetative

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cell growth and biomass accumulation. At the second phase (the ‘induction’ or ‘red’ stage), accumulation of astaxanthin is induced in the green cells by stressing them with high light, nitrogen and/or phosphorous shortage [20]. As a result, the cells become red

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due to the high astaxanthin content.

Previously we demonstrated that the analysis of CF reveals a great deal of

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valuable information about the stress acclimation and condition of H. pluvialis cells during their reversible transition between the green and the red stage [16]. Moreover, the analysis of CF transient is applicable for the monitoring of H. pluvialis in case of its

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laboratory [21] and outdoor [18,22] cultivation.

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As mentioned above, CO2 enrichment is a pre-requisite for achieving high microalgal culture productivity. At the same time, high CO2 levels are often stressful to microalgae, especially for their photosynthetic apparatus [6,24]. Recent studies of the

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cultivation of H. pluvialis under elevated CO2 demonstrated that increasing of CO2 percentage in the gas mixture used for the culture sparging to 5% are beneficial for

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astaxanthin accumulation [23,25]. Mutants of H. pluvialis with enhanced growth and astaxanthin accumulation under 6% [25] and 15% CO2 enrichment [26] were obtained by

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Co γ-irradiation. These mutants characterized by the low-pH resistance [25] and

up-regulation of the pyruvate pathway and the pathways of carotenoid and fatty acid biosynthesis [27]. Moreover, sequential photobioreactor allowed direct injection of flue gas for CO2-enrichment of H. pluvialis culture [28]. However, as far as we know, the literature lacks detailed reports on the evolution of primary photochemistry as revealed by the CF analysis and verified by direct carbon content measurements. To bridge this gap, at least in part, we followed the changes in

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primary photochemistry of H. pluvialis via recording the CF transients (OJIP-curves) [8–13] and the induction of regulated non-photochemical quenching [13] of the excited chlorophyll states. We also evaluated the relationships of these parameters with growth rate, astaxanthin accumulation and directly assayed carbon content of the H. pluvialis biomass under different CO2 levels. 2. Materials and methods

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2.1. Strain and cultivation conditions

The Haematococcus pluvialis strain IPPAS H-2018 (former BM1) from the

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White Sea [29] served as the object in the present work. The cultures were initiated

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using the cells from exponentially growing pre-cultures at a density of 15 mg of total chlorophyll (Chl) per 1 L of suspension. The algae were cultivated in 0.6 L glass

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columns (40 mm ID) in 400 mL of BG-11 medium [30] at 25 °C under continuous illumination by cold white LED lamps. Pilot experiments with vegetative cell cultures were carried out at a photon flux density (PFD) of 40 µmol (quantum) /m2/s, as in our

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previous works with H. pluvialis [14,29]. However, the elevated CO2 of 5% did not cause a statistically significant change in the culture parameters as compared to the

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atmospheric CO2 level. The lack of the effect in this case might stem from light limitation and hence from energy shortage for CO2 assimilation. In the following

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experiments, the irradiance was increased to 60 µmol (quantum) /m2/s. The cultures were sparged (1 vvm) with atmospheric air (0.04% CO2) or air-gas mixture (AGM)

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containing 5%, 10% or 20% CO2 (v/v). For the induction of astaxanthin accumulation, the cells were incubated in the BG-11 medium lacking KNO3 (BG-110) [31] at a high PFD (480 μmol (quantum) /m2/s;

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high light, HL); the other conditions were the same as specified above. 2.2.Growth parameters Culture growth was monitored via pigment and biomass accumulation. Pigments were extracted with dimethyl sulfoxide as described elsewhere [29]. Total carotenoid (Car),

Chl

a

and

Chl

b

concentrations

in

the

extracts

were

assayed

spectrophotometrically [32]. Astaxanthin content was measured as described in [33]. Dry weight (DW) was determined gravimetrically [34]. 2.3. Determination of C and N content of and CO2 capture by the H. pluvialis cells

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For elemental C and N determination, H. pluvialis cells were taken at the 6th day (vegetative growth, exponential phase), 13th day (vegetative growth, stationary phase) of culturing and after 5 days of the stress exposure (red cells). Elemental carbon and nitrogen content in the biomass was determined using a Vario EL Cube CNS (carbon, nitrogen and sulfur) element analyzer (Elementar, Germany) calibrated with a certified acetanilide

standard

(Elementar,

Germany)

using

C/N

according

to

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manufacturer’s protocol.

mode

2.4. Chlorophyll a fluorescence analysis

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CF in dark-adapted cells was measured with Fluorpen FP100s portable Pulse

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Amplitude Modulated fluorometer (Photon Systems Instruments, Czech Republic) as described in [16]. Stern-Volmer NPQ parameter was determined by the manufacturers

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protocol in the cells exposed to the actinic light for 60 s. During the actinic light exposure, five recordings of maximum CF intensity in the light-adapted state (Fm') were made after saturation flashes starting after seven seconds of the actinic light exposure

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and repeating at 12 s intervals. 2.5. Light microscopy

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The cells were studied in bright field or fluorescence mode under Eclipse 90i (Nikon,

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Japan) motorized photomicroscope. The cell viability was estimated by the presence of the red CF [35]. The cells lacking Chl red fluorescence were considered as dead. 2.6. Data treatment

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All measurements were carried out in two biological and three analytical replicas. Data were treated with OriginPro 8 software (OriginLab Corporation, USA).

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Average values and corresponding standard deviations are presented. Significance of the mean difference was tested using the standard t- and F-tests (P = 0.95). 3. Theory and calculations 3.1.CF parameters

The calculation of PS II primary photochemistry parameters from CF transient curves was carried out following the approach of Strasser et al. [12]. The CF transient curves recorded in the dark-adapted samples (Fig. 1) are characterized by the presence of the initial point O, where the CF intensity (Fo) is at its minimum, the inflections (J

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and I) and the point P, where the CF intensity is maximal (Fm). The maximal PS II photochemical quantum yield φ(PS II)0 [8-10,12] was estimated as φ(PS II)0 =

Fv , Fm

where Fv ≡ Fm − Fo is the variable fluorescence. The probability (Ψ0 ) of electron

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transport beyond primary PSII quinone acceptor (QA) during the time of CF rising to Fm [12] was estimated as

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Ψ0 = 1 − VJ ,

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where FJ is the fluorescence intensity at point J, VJ ≡ (FJ − Fo)⁄Fv is the relative height of the OJ-step. Fraction of the energy for multiple events of QA reduction during the

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time of fluorescence rise to Fm is proportional to the normalized area above the OJIPcurve (Sm) [12]:

tm

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tm

1 Sm = ∫ (Fm − F(τ))dτ = ∫ (1 − V(τ))dτ, Fv 0 μs

0 μs

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where V(τ) ≡ (F(τ) − Fo)⁄Fv is the relative variable fluorescence. The primary quinone acceptor turnover number [12] (the number of QA red/ox events during the time

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of fluorescence rise to Fm), N(QA ), was estimated as the ratio of N(QA ) =

Sm , Ss

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where Ss is the normalized area above OJIP-curve in the case of single QA reduction event. Assuming exponential initial slope of the OJIP-curve [12,36], Ss is inversely proportional to the first derivative of V at the origin of the curve (Mo = (dV⁄dt)t=0 μs ) divided by VJ : −1

Ss = (Mo/VJ ) . The derivate Mo was calculated as the ratio of the increments of the normalized variable fluorescence and time between 50 and 300 µs [12]:

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dV V(300μs) − V(50μs) ( ) ≅ . dt t=0 μs 250μs The intensity of non-photochemical quenching of excited chlorophyll states was estimated as the Stern-Volmer coefficient NPQ [8,9,13]: Fm − Fm′ , Fm′

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NPQ =

where Fm and Fm′ is the maximal intensity of CF of dark-adapted and light-adapted

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cells respectively. The highest NPQ values presented on the figures were registered

3.2. Estimation of the CO2 capture capability

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according to the Fluorpen FP100s manufacturer’s protocol.

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To estimate the capability of the H. pluvialis cells to capture CO2 from the AGM, the average daily removal of CO2, AR(CO2), was calculated based on the

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elemental carbon (C) content in the biomass and DW accumulation. The latter was calculated as

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ΔDW = DWf – DW0 ,

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where DW0 is the initial DW and DWf is the DW at the end of the experiment. Assuming that the mass fraction of carbon, ω(C), in the biomass did not decline during cultivation, the amount of C assimilated by one liter of the vegetative cell culture can be

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calculated as

𝐦(𝐂) = 𝛚(𝐂)𝐞𝐱𝐩 ∆𝐃𝐖𝐞𝐱𝐩 + 𝛚(𝐂)𝐬𝐭𝐚𝐭 ∆𝐃𝐖𝐬𝐭𝐚𝐭 ,

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where ω(C)exp, ΔDWexp and ω(C)stat, ΔDWstat are the biomass accumulation and carbon mass fraction at the exponential and stationary growth phases, respectively. Similarly, the amount of C assimilated during the stress exposure for astaxanthin accumulation induction was determined using corresponded DW accumulation and C content values, ΔDWstat and ω(C)ind. The total volumetric removal (R) of CO2 per liter of the culture is then R(CO2 ) = m(C)

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M(CO2 ) , M(C)

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where M(CO2) and M(C) are molar weights of CO2 and C, respectively. The specific AR per the unit of cultivation time, t, would be AR(CO2 ) =

R(CO2 ) , Δt

where Δt is the time of cultivation.

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4. Results and discussion 4.1.The analysis of CF transients confirms the beneficial effect of the moderately high

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CO2 level on the H. pluvialis cells

To study the effect of the elevated CO2 levels on photosynthetic activity of H.

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pluvialis cells and its possible relationships with astaxanthin accumulation under the stressful conditions, the CF transient (OJIP-curves) (Fig. 1) recorded at different CO2

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levels was analyzed and the parameter φ(PS II)0 which is widely used for the assessment of the potential efficiency of the primary processes of photosynthesis was

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calculated [8–12]. H. plivialis OJIP-curve shape was typical for microalgae containing the J and I inflections; the fluorescence decayed after the maximum P reflecting the photochemical utilization or thermal dissipation of the absorbed light energy [21,37].

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The response of H. pluvialis cells to the stress conditions was accompanied by a

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decrease of the OJIP-curve amplitude (Fig. 1). The vegetative cell cultures sparged with the air-gas mixture (AGM) containing less than 20% CO2 possessed φ(PS II)0 in the range of 0.65–0.77 (Fig. 2A), which is

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typical for H. pluvialis cells [16,18,21,22,38] suggesting that CO2 in this concentration range was not deleterious for the PSA of the microalga. By contrast, the addition of

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20% CO2 to the AGM resulted in a sharp decline of φ(PSII)0 (to 0.55±0.01, Fig. 2A) manifesting the pronounced stress to the vegetative cells. Under the conditions conductive for accumulation of astaxanthin, the stress-induced drop of φ(PS II)0 was sharper in the case of sparging with 10% or 20% CO2 (0.27±0.01 at 3 day and 0.31±0.01 at 2 day respectively). Generally, unfavorable conditions trigger the rapid closure of reaction centers in H. pluvialis cells [18], which is manifested by φ(PS II)0 decline. At the same time, photosynthetic activity is normally retained in this microalga to a certain degree, at least for several days of stress exposure [16,18,38]. We compared the retention of the

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photosynthetic activity under the stressful conditions as a function of the CO2 level in the AGM. As shown in Fig. 2B, the retention of the photosynthetic activity was most significant in the cultures sparged with 5% CO2. In this case, 80.1% of the initial φ(PS II)0 was retained at the 3rd day of the stress exposure, whereas a lower retention of the photosynthetic activity was documented for the control, 10% and 20% CO2-sparged cells (69.4%, 53.0% and 43.5%, respectively). Remarkably, addition of 5% CO2 to

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AGM prevented a sharp decline in Chl in the stressed cells (27.4 % of the initial Chl content was retained whereas in the air-sparged control the Chl retention was as low as

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12.6 %; Fig. 4D). It is possible to speculate that the slower Chl decline under

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moderately high CO2 manifested the longer retention of the H. pluvialis PSA activity which might supply more energy and reducing power for carbon assimilation and, eventually, to the astaxanthin biosynthesis. Taking into account the simultaneous

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retention of Chl, one may think that the 5% CO2-sparged cultures indeed retained their photosynthetic activity for a longer period, probably due to a higher sink capacity of the

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cell in the absence of inorganic carbon limitation.

The widely used φ(PS II)0 parameter reflects the potential efficiency of charge separation in the PS II reaction centers in the dark-adapted state. However, in some

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cases its sensitivity can be impaired [10–13,39] disturbing correlation of the actual CO2

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assimilation rate and φ(PS II)0 [10,39,40]. Such situations call for additional CF parameters to be employed for a more reliable assessment of the phototrophic cell condition. In particular, the redox state of the plastoquinone pool should be taken into

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account [40–43]. Towards this end, we analyzed additional CF parameters reflecting the capability of the chloroplast ETC of intersystem electron transfer.

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In the exponentially growing vegetative cell cultures, an increase in CO2 to 5% was accompanied by a Ψ0 rise from 0.46±0.01 to 0.50±0.01 during the first three days of the cultivation (Fig. 2C) evidently manifesting the up-regulation of the photosynthesis. By contrast, the CO2 concentrations above 5% lead to a decrease in Ψ0 which was especially pronounced under 20% CO2 (Fig. 2C). Importantly, the induction of astaxanthin biosynthesis by the HL and N-starvation was accompanied by Ψ0 decrease but this effect was less profound in the 5% CO2-sparged culture (Fig. 2C). On the contrary, under 20% CO2, it dropped nearly to zero at the first day of cultivation time suggesting the cessation of the photosynthetic carbon fixation (Fig. 2C).

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Thus, the low values of the probability of electron transport beyond the primary PSII quinone acceptor QA (Ψ0) manifested that ETC was overreduced. This situation was a common result of the impaired dark reactions of photosynthesis and/or other metabolic pathways [12,40,41,43]. Such situations were characterized by elevated risk of toxic reactive oxygen species formation [44]. On the contrary, high Ψ0 values reflected the efficient light energy utilization [40,41,43].

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It was found recently that the reorganization of H. pluvialis cell during stressinduced formation of the astaxanthin-rich resting cysts (haematocysts) is accompanied

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by the vigorous functioning of the chloroplast ETC [16,18] and the elevated turnover of

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primary PS II quinone acceptor(QA), N(QA) [16]. The high N(QA) values should reflect a high rate of the photosynthetic electron transport [40,41]. Together with φ(PSII)0, high values of these parameters are characteristic of cells with high PSA activity. On the

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contrary, in the cells with impaired photosynthesis N(QA) declines sharply. Indeed, the transformation of vegetative cell to haematocyst induced by HL and N starvation is

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accompanied by a transient sharp increase in N(QA) [16]. The highest N(QA) was recorded at the 4th day under inductive conditions in the cultures sparged with 5% CO2 (2500±400 vs. 1300±500 in the air-sparged cultures; Fig. 3B). In the cultures under 10%

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CO2 the N(QA) transient maximum was lower and occurred sooner than under 5% CO2

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(Fig. 3B). A similar response was observed in higher plants e.g. Lolium perenne subjected to salt stress displaying a short-term increase in N(QA) [43]. Accordingly, the transient up-regulation of metabolism seems to be a widespread stress response in

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photoautotroph cells.

The electron flow through the chloroplast ETC is regulated, in particular, by the

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reduction state of the plastoquinone pool (PQ) [45]. Judging from the data obtained in this work, the increase of CO2 to 5% in the AGM relieved the carbon limitation of the dark reactions of photosynthesis increasing their electron sink capacity and delaying the reduction of PQ apparent as a decline in N(QA) and Ψ0. Obviously, it was not the case in the 10% CO2-sparged cultures. In this case, the stress imposed by a very high CO2 level presumably inhibited the carbon assimilation resulting in the fast overreduction of the electron carriers in chloroplast ETC. This might explain an earlier onset of the N(QA) decline in comparison with the air-sparged cultures (Fig. 3B).

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The increase of CO2 level in the AGM to 20% led to a rapid monotonous N(QA) decline (Fig. 3B). This effect might stem from the disappearance of the thylakoid transmembrane proton gradient (ΔpH) as well as strong inhibition of Calvin-Benson cycle enzymes. In this case, an abrupt inhibition of chloroplast ETC and a general disturbance of the cell metabolism might even block its transformation to haematocyst. The inability of the resting cell formation was often fatal under the stressful conditions

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employed in our work resulting in high cell mortality (Fig. S1).

4.2. The retention of photosynthetic activity under the stress is accompanied by the

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adequate protection by non-photochemical quenching of the excited states of

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chlorophyll

In general, photoautotrophs adjust photosynthetic carbon fixation by regulation

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of gas exchange (in the case of higher plants), reorganization of photosynthetic apparatus (its light harvesting antenna) and subsequent redistribution of the absorbed energy between thermal dissipation and photochemical pathways [9,10,39,41,42].

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Non-photochemical quenching of the excited states of Chl is essential for safe dissipation of the light energy absorbed in excess and protection of the cell against

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photodamage, especially under stress [13,16]. Since non-photochemical quenching might play an important role in the stress tolerance of H. pluvialis [16], we followed the

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changes in the NPQ parameter in the course of the alga cultivation. High NPQ values suggest high intensity of the processes of protective regulated energy dissipation, NPQ increasing is an adequate response of the cell to stress conditions essential for its

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viability [13]. Impairment of the cell ability to build-up non-photochemical quenching increases the risk of photooxidative damage to and, ultimately, death of the cell

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[13,37,42]. At the same time, unwarranted high non-photochemical quenching is deteriorative for CO2 assimilation and hence for the culture productivity provided that it is not limited by inorganic carbon [37, 42]. Therefore, responsiveness of NPQ regulation is essential for balancing of the photoautotrophic cell safety vs. satisfying its energy demand. In the 5% CO2-sparged cultures NPQ was lower than in the control during exponential and early stationary growth phases (0.55±0.07 vs. 1.00±0.01, respectively, at the 3rd day of cultivation; Fig. 4C). Since the light energy absorbed by the cell is either utilized photochemically (mainly for C fixation) or non-photochemically

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dissipated (as heat or luminescence) [8,9,10,12,13], a low NPQ level in the exponentially growing cultures likely manifested efficient photochemistry and, eventually, carbon fixation. In general, low NPQ values are typical for vigorously dividing microalgal cultures and rapidly growing young plants with a high energy demand [18,41]. Thus, low NPQ values under 5% CO2 enrichment suggest the elevated assimilatory capacity of the cell with regard to inorganic carbon.

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Under the stressful conditions, the highest NPQ was observed in 5% CO2sparged H. pluvialis cultures (compared with 10%, 20% and air control). Moreover, in

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this case, high NPQ values were retained for a longer time (till the 5th day of cultivation;

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Fig. 4D). It was essential for protection of the cells with high photosynthetic activity against photodamage. In 5% CO2-sparged cultures as well as in the air-sparged control NPQ showed a bi-phasic kinetics. The increase in NPQ was followed by its sharp

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decline. By contrast, a low NPQ showing no rise was recorded in the 10% and 20% CO2-sparged cultures (Fig. 4D).

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Under an abrupt stress, the cell might not be able to execute an adequate protective response resulting in elevated cell mortality. As was described previously [37], a severe temperature stress caused much weaker NPQ response in comparison

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with a moderate temperature stress. A similar situation might took place in the H.

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pluvialis cells under the highest CO2 enrichment under our experimental conditions. Taking into account the low Ψ0 and φ(PS II)0 values recorded in these cultures (Fig. 3), it was possible that the ΔpH-dependent NPQ mechanisms (e.g. violaxanthin cycle) were

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somehow impaired in this case due to the stroma acidification [13,16]. NPQ is thought to operate in the cell in the beginning of its transformation to haematocyst [16] when its

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metabolic activity is still high [16,18]. This suggestion is compatible with the high NPQ and N(QA) values recorded within the short period after the stress exposure [16]. Indeed, the stressed H. pluvialis cultures retained its photosynthetic activity for a certain period characterized by a high NPQ serving presumably for the protection of PSA (Fig. 4D). Later, upon accumulation of large amounts of astaxanthin it decreases nearly to zero [16]. In view of these findings, a sizeable NPQ could be regarded as a manifestation of the residual photosynthetical activity in the stressed H. pluvialis cells. 4.3. H. pluvialis growth and astaxanthin accumulation at different CO2 levels

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The increase in CO2 concentration in the AGM to 5% improved the biomass accumulation (3.4±0.2 g/L DW vs. 0.4±0.1 g/L DW) (Fig. 1A) and Chl content in the vegetative cultures in comparison with the air-sparged control (1.5±0.1 mg/L vs. 8.4±2.5 mg/L) (Fig. 1C). To assess the degree of stress of the H. pluvialis cultures under our experimental conditions, we also monitored their carotenoid-to-chlorophyll (Car/Chl) ratio (Fig. 1B)

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as a sensitive stress marker in the microalgae [32]. Under the stress conditions, Car/Chl ratio increased sharply manifesting the onset of the stress. The addition of 5% CO2 to

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the AGM did not result in a significant increase in Car/Chl ratio in comparison with the

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air-sparged control (Fig. 1B) suggesting that the moderately high CO2 level in the AGM did not exert deteriorative effects on the H. pluvialis vegetative cells. Moreover, the cultivation with 5% СО2 was accompanied by a massive formation of zoospores (Fig.

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S2), which are the most metabolically active and vigorously dividing cells in H. pluvialis culture [46]. It is likely that the increase in the external CO2 by more than two

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orders of magnitude (5%) relieved the inorganic carbon limitation of the photosynthesis in the H. pluvialis cells taking place at the atmospheric (0.04%) CO2 level resulting in a sizeable increase of the DW accumulation.

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By contrast, a further increase in СО2 concentration in the AGM to 10% resulted

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in the culture growth rate decline (Fig. 1A,C). In this case, the maximal Chl content was 42.4±5.0 mg/L and Car content was as high as 31.9±1.5 mg/L yielding the Car/Chl ratio mass ratio of 0.9±0.2 by the 13th day vs. 0.4±0.1 in the air-sparged control (Fig. 1). This

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finding suggested that sparging with the AGM containing 10% СО2 under our experimental conditions imposed a considerable stress to the culture. In the cultures

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sparged with the AGM containing 20% СО2, the deteriorative effect of the very high carbon dioxide level was manifested by a dramatic increase of Car/Chl ratio (from 0.3±0.1 to 0.9±0.1 within 6 days) and the cessation of DW and Chl accumulation (Fig. 1). The adverse effects of the very high CO2 levels might be mediated by disturbing of the cell pH homeostasis [4,6,25]. This process leads to acidification of the cytoplasm and chloroplast stroma [6,25] resulting in the inhibition of Calvin-Benson cycle enzymes [6] impairing the assimilatory capacity of the cell. In addition, cytoplasm acidification might lead to the inhibition of glycolysis enzymes (particularly, phosphofructokinase),

14

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protein and nucleic acid synthesis as well as disturbance of the cytoskeleton formation [47]. In our preliminary experiments with the H. pluvialis, the accumulation of astaxanthin was induced by an increase in irradiance to 480 µmol (quantum) /m2/s and transferring of the cells to distilled water [29]. However, sparging with the AGM enriched with CO2

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under these conditions led to mass cell mortality (Fig. S2) and a dramatic decline in astaxanthin accumulation (data not shown). As in the vegetative cells, the deteriorative

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effect of the very high CO2 levels stems likely from the cytoplasm acidification. Replacement of distilled water with BG-110 medium at the ‘red’ phase declined the cell

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mortality probably due to an increased buffer capacity of the cells in the presence of + + + + metal ions facilitating the pumping of protons out of the cell by Na /H and K /H

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antiporters maintaining the cell pH homeostasis [47,48].

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As shown in Fig. 4D, biosynthesis of astaxanthin was not, in principle, inhibited at CO2 concentrations up to 20% albeit the latter brought about a high cell mortality (Fig. S2). Indeed, the maximum Car content was observed in the culture sparged by 5%

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CO2 (3.7 % DW vs 2.1 % DW in the control, 2.3%; 1.4% under 10% and 20%

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respectively, Fig. 4D). The beneficial effect of the moderately high CO2 level under the stress conditions was also described by Kang et al. [23] and Cheng et al. [25] although the maximum astaxanthin level attained in their experiments was approximately one

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order of magnitude lower.

4.4.A moderate elevation of CO2 in the AGM facilitates the assimilation of carbon by H.

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pluvialis cells

The stoichiometry of C and N is an important marker of microalgal physiological condition: normally, the C/N ratio is lower in rapidly dividing cells since they contain more N-rich compounds e.g. proteins, nucleic acids and Chl which are essential for their active metabolism than the stationary-phase cells [49,50]. Moreover, under the limitation by inorganic C, the C/N ratio of microalgal cells which are not otherwise limited is expected to be proportional to the CO2 level, whereas lifting of this limitation disturbs this correlation [50]. Thus, the analysis of the C/N as function of CO2 content in the AGM may be useful to believe the C-limitation.

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The lowest C/N values typical of green algae [49,51] were recorded at the exponential growth phase (5.4±0.5 g/g – 6.9±0.1 g/g; table 1) when a significant part of the cell dry weight was comprised photosynthetic pigments and proteins (reviewed in [52]). Upon the onset of the stationary growth phase, the cell division rate slowed down giving rise to their C/N ratio. Under the stressful conditions, the 5% CO2-sparged cultures demonstrated a sharp rise of the C/N ratio to 42.8 ± 1.0 g/g (table 1) which is,

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as far we know, among the highest values for algae [49,51]. The ability of H. pluvialis to change its elemental composition in a wide range in the response to changing in the

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cultivation condition was likely a manifestation of its remarkable physiological

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plasticity. Notably, the 5% CO2-sparged cultures possessed a C/N ratio higher than that of the air-sparged cultures (table 1) but a further increase of CO2 in the AGM to 10% or 20% did not lead to an increase in C/N ratio (table 1). In view of this, the atmospheric

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CO2 concentration was indeed limiting for H. pluvialis under our experimental conditions and this limitation was relieved by the increase of CO2 level in the AGM to

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5%.

To estimate the potential efficiency of CO2 capture of the H. pluvialis cultures, we measured the elemental carbon content in the H. pluvialis biomass as a function of

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cultivation stage and CO2 concentration in the AGM. The highest volumetric CO2

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removal, AR (371.1±41.9 mg/L/day or 0.66% of total CO2 injected), was shown by the vegetative cells H. pluvialis sparged with 5% of CO2, this was in agreement with the data obtained for other microalgae [6,53], reviewed in [5]. In the case of control AR was

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207.5±70.5 mg/L/day (45.9% of total CO2). The increase of CO2 in AGM to 10% and 20% led to a decline in the CO2 removal likely due to the negative effect on the

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photosynthetic CO2 fixation by the cells. In general, acclimation of photosynthetic cells to stress conditions accompanied by carbon assimilation decline [9,10,40,54]. However, initial steps of stress response in higher plants in some cases characterized by the retention of photosynthetic activity and CO2 fixation [40,43]. The same situation was observed in H. pluvialis cells under the conditions of astaxanthin synthesis induction in the case of air and 5% CO2 (table 1). The short-term period of increased assimilatory capacity might be essential for the acclimation to new conditions. In contrast to higher plants adjusting the inflow of CO2 by regulating their epidermal (stomatal) conductance [40,42], microalgal cells are more

16

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vulnerable to the deteriorative effects of high CO2 concentrations. Accordingly, the intracellular

protective

mechanisms

(e.g.

non-photochemical

quenching,

pH

maintenance and modulation of CO2 concentration mechanisms) are more important for unicellular algae [6,24,25,47,48]. Expectedly, the CO2 removal was higher under the conditions conductive for vegetative cell growth, likely due to the more efficient photosynthesis and carbon

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assimilation. The percentage of CO2 capture for typical Chlorophyta (Scenedesmus, Chlorella, Tetraselmis, Chlamydomonas) is in the range of 34.2% to 97% under the air

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(0.04% CO2) [4] and from 0.3% to 5.3% under a higher CO2 level [4], as was reviewed

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in [3]. The highest rate of CO2 capture (approximately 0.37 g or 0.19 g per liter of the culture volume per the day) was attained by the exponentially growing vegetative cell culture continuously sparged with 5% CO2. It is difficult to compare these data for

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different strains, because they strongly depend on the culture growth conditions as well as photobioreactor design [3,4]. In addition, there is a discrepancy in the methods of the

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culture productivity estimation as a function of CO2 level. Thus, the results based on cell count [3], DW determination [53] and measurements of optical density [4,23] are hardly comparable. Nevertheless, a common trend is comprised by a sharp decrease of CO2

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assimilation by the cultures when its volumetric percentage exceeds 5%, the effects of

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further CO2 increase in its assimilation are less pronounced. The existence of a such threshold might indicate the relief of the inorganic carbon limitation evident under the atmospheric concentration and 5% CO2.

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Finally, leveraging the full biotechnological potential of microalgae requires the knowledge of their optimum CO2 range (according to our present data, around 5% for

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the wild type strain). It is also significant that the sparging with the elevated CO2 is beneficial not only during the vegetative growth of H. pluvialis, but also in the beginning of the haematocyst formation (albeit the CO2 removal by the red cells is lower than that by the green cells). 5. Conclusion Effects of CO2 enrichment on the primary photochemistry, engagement of photoprotective mechanisms and carbon fixation capacity in the carotenogenic microalga H. pluvialis IPPAS H-2018 depends on the rate of CO2 injection (CO2 concentration in the AGM). Moderate CO2 levels were beneficial since they obviously

17

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lift the inorganic carbon limitation favoring the carbon fixation, biomass and astaxanthin accumulation. By contrast, the very high levels (10% and higher) cause too severe a stress which impaired photosynthesis and hence was deleterious for growth and astaxanthin accumulation. The CF-derived parameters used in this work turned to be suitable for monitoring of the physiological condition and photosynthetic activity of the CO2-enriched cultures of H. pluvialis. Collectively, the results obtained in this work

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support, in principle, the possibility of H. pluvialis cultivation at a moderately high CO2 levels for the combined of the astaxanthin production and CO2 bio-sequestration.

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Finally, a further increase in the efficiency of the combined process will require

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additional optimization of H. pluvialis cultivation and CO2 injection. This effort would also benefit from continuing studies of the mechanisms of CO2 tolerance of and assimilation by the microalgal cells.

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Acknowledgements

The funding by Russian Science Foundation (grant 14-50-00029, microalgae

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cultivation) and Russian Foundation for Basic Research (grant 15-04-01061, element

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composition assay) is gratefully acknowledged.

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Figure captions Fig. 1. Chlorophyll fluorescence transient (OJIP) curves of the H. pluvialis under the vegetative growth conditions (A) and under the stress (nitrogen deprivation, high light) (B).

Fig. 2. The changes in (A, B) φ(PSII)0 and (C, D) Ψ0 in the course of vegetative growth

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(A, C) and stress exposure (nitrogen sufficient and the high light) (B, D) of H. pluvialis.

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The CO2 level in the AGM used for the culture sparging is indicated in the panel A.

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Fig. 3. The kinetics of QA turnover number (A, B) and NPQ (C, D) in the vegetative (A, C) and stressed (by nitrogen deprivation and high light) (B, D) cells of H. pluvialis. The

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level of CO2 in the AGM used for sparging of the cultures is indicated in the panels.

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Fig. 4. The kinetics of H. pluvialis culture dry weight (A), Car/Chl ratio (B) under the conditions of vegetative growth. Total chlorophyll content in the vegetative (C) and stressed (by nitrogen deprivation and high light, D) cell cultures conditions (see

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Methods). Insert: total carotenoid content of the H. pluvialis cultures grown at different

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CO2 level after 7 day of the stress exposure. The CO2 level in the air-gas mixture used

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for the culture sparging is indicated in the figures.

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Tables Table 1. Elemental C and N contents and their mass ratio in the biomass of H. pluvialis from different growth stages and average daily removal of CO2 by the H. pluvialis cultures sparged with AGM containing different CO2 concentrations. Average values and standard were presented. There were total of 6 replications. Significantly different values are labelled with different letters. Element content, % DW N C

C/N, wt/wt

Vegetative (green) cells

1.3±0.2a

Stationary, 3 d

6.1±0.2b

45.7±2.7a

7.5±0.3b

0.3±0.1b

Exponential, 11 d

7.8±0.1a

46.7 ± 0.2a

6.0±0.1c

2.6±0.2c

Stationary, 2 d

4.5±0.3c

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d

0.3

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10%

D

Exponential, 8 d

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5.4±0.5a

7.3 ±

50.6±1.7b

49.4±0.4b

d

0.2±0.1b

6.8±0.2e

1.4±0.2a

5.8±0.1e

47.5±1.3b

8.2±0.1f

0.2±0.1b

Stationary, 6 d

7.1±0.1d

48.9±1.1b

6.9±0.1e

n/d

5%

7d

1.2 ± 0.1f

49.1±1.0b

10%

7d

2.2 ± 0.2f

AC 7d

1.35 ± 7d

b

221.9±43.6

n/d*

Stressed (red) cells 50.9±2.3b

20%

12.1±0.5

a

1.5±0.1f

(air)

a

371.1±41.9

Stationary,5 d

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Control

207.5±70.5

46.1±1.1a

5%

20%

𝐴𝑅(𝐶𝑂2 ), mg/L/day

8.7±1.1a

Control Exponential, 10 d (air)

∆𝐷𝑊, g/L

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Growth phase (day of cultivation)

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СО2 vol. %

0.04

f

34.2±0.4 g

42.8±1.0

0.4±0.2b

106.6±58.1 c

128.6±54.1

h

0.5±0.2b

55.0±0.4c

24.8±2.1i

0.2±0.1b

n/d

50.5±0.9b

37.4±1.8j

0.2±0.1b

n/d

26

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*

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not detected

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Figure 4

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Highlights

The effect of CO2 on light energy utilization in Haematococcus cells was evaluated.  The moderately high CO2 level promotes photosynthesis and carbon assimilation.  Enhanced CO2 assimilation accompanied by NPQ downregulation.  The induction of astaxanthin accumulation is accompanied by an upsurge of C/N ratio.

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