Culture Conditions and Experimental Set-Up
Emiliania huxleyi (Lohmann) Hay et Mohler (RCC 1226) cells were provided by the Roscoff Culture Collection, France. This is a heavily calcified type A strain isolated from the Atlantic Ocean close to the Norwegian coast in July 1998. Cells were grown in 3 L sterile Perspex cylinders (Plexiglas XT® 29,080) in artificial seawater medium [34] enriched with f/2 nutrients [35]. Cultures were grown at 16 °C, 120 µmol photons m−2 s−1 photosynthetic active radiation (PAR, 400–700 nm), and at a 14:10 h light: darkness photoperiod. Irradiance was provided by fluorescent tubes of daylight type Osram Sylvania standard truelight 18 W, as measured by a submersible micro quantum sensor (US-SQS/L, Walz, Germany) connected to a Li-COR 250A radiometer. Cells were exposed to low pH (7.7, LP) by either aerating with air enriched with CO2 to 1200 µatm (HC-LP, high carbon and low pH) or by HCl additions in combination with non-enriched aeration (LC-LP, low carbon and low pH). The control condition consisted of non-acidified, non-enriched cultures at 400 µatm CO2 (LC-HP, low carbon and high pH). All treatments, including the control, consisted of triplicate cultures continuously stirred with a magnetic bar and aerated to ensure homogeneity without mechanical stress and to avoid cell shading. HC levels of CO2 were obtained by mixing atmospheric air with pure CO2 (Biogon®, Linde, Germany) to achieve 1200 µatm pCO2 inlet flow in each culture was measured by non-dispersive infrared analysis by using a CO2 gas analyser (LI-820, Li-COR) at 200 mL min−1. The air was filtered through Millipore 0.2 µm fiberglass filters (Merck, Germany). For lowering the pH with HCl, the required volume of 12 N HCl (Merck, Germany) was added to the culture medium until pH = 7.7 was reached prior to cells inoculation. The medium and stock cultures were pre-acclimated to the different pCO2 and pH conditions for 72 h in order to avoid transient effects [36] under the conditions described just above. The experiment lasted 9 days, and the 3 L cultures were sampled by extracting 175 mL on days 2, 4, 7, and at the end of the experiment, thus the remaining volume before last sampling was 82.5% of the initial.
Carbonate System (pCO2, DIC, Ωcalcite, pH, Alkalinity)
pCO2, DIC, and Ωcalcite were calculated from daily measurements of pH, temperature, salinity, and total alkalinity (TA) using the CO2Calc software [37] fitted using GEOSECS constants. pH was measured in all the cultures by using a pH-meter (CRISON Basic 20 +) calibrated daily using the NBS scale. Salinity was measured with a conductivity meter (CRISON 524). The accuracy of the pH-meter and conductivity meter were ± 0.01 pH units and ± 1.5%, respectively. TA was measured using the Gran’s potentiometric method [38]. No certified reference standards were used.
Cell Abundance and Growth Rates
Cell density was determined using an Accuri™ C6 flow cytometer (BD Biosciences, USA) equipped with an air-cooled laser providing 15 mW at 488 nm and with a standard filter set-up by using 1 mL samples. The trigger was set on red fluorescence and samples were analysed for 90 s at an average flow rate of 14 μL min−1. Cells were discriminated on the basis of the side-scatter signal (SSC) versus chlorophyll [39, 40].
The growth rate (µ) was calculated by fitting the cell density data to the logistic growth model (Eq. 1):
$$Ln\left(\frac{\left(K-N\right)}{N}\right)=Ln\left(\left(K-{N}_{0}\right)-1\right)-\mu t$$
(1)
where K refers to the loading capacity, N is the cell density at any given time, N0 is the cell density at time 0, µ is the intrinsic growth rate, and t is the time (in days). The logistic model was preferred over the exponential model because the cultures reached the stationary phase.
Chlorophyll a (Chl a) Concentration and In Vivo Chl a Fluorescence Associated to PSII
Samples of 5 mL were collected from each culture, centrifuged at 4000 g, and the pellet snap frozen in liquid nitrogen and kept at − 80 °C until analysis. Pellets were incubated overnight at 4 °C in N,N-dimethylformamide (Sigma-Aldrich, USA) for Chl a extraction. The concentration was determined spectrophotometrically and calculated accordingly [41].
The optimal quantum yield (FV/Fm) for charge separation in photosystem II (PSII) is frequently used as an indicator of photoinhibition, reflecting the general status. In vivo chlorophyll a fluorescence of PSII was determined by using a pulse amplitude modulated fluorometer Water-PAM (Heinz Waltz, Effeltrich, Germany) as described by Schreiber et al. [42]. F0 and Fm were determined in 10-min dark-adapted freshly taken 2 mL culture samples, to ensure oxidation of primary quinone acceptor (QA), to obtain the FV/Fm. FV is the variable fluorescence of dark-adapted algae when all the reaction centres are opened as Fm − F0. Fm is the maximal fluorescence intensity with all PSII reaction centres closed obtained after an intense actinic saturation light pulse > 4000 µmol photons m−2 s−1, and F0 is the basal fluorescence (minimal fluorescence) of dark-adapted after 10 min. Using the software WinControl-3.25, rapid light curves (RLCs) were constructed and fitted to the nonlinear least-squares regression model of Eilers and Peeters [43] to obtain the initial slope of the curve related to the photosynthetic light-harvesting efficiency (αETR) (as an estimator of photosynthetic efficiency) and the relative maximal electron transport rate (rETRmax). The actinic light intensities were selected according to the saturation pattern and measured in the PAM cuvette using a US-SQS/L micro quantum sensor (Walz) attached to a Licor 250-A radiometer. The light requirement for saturating photosynthetic rate (Ek) and the maximum irradiance before photoinhibition of rETR was observed (Eopt) were derived from rETRmax and α and ß slopes, respectively, where ß is the slope of rETR decay at high irradiance.
Cell Viability and Reactive Oxygen Species (ROS)
Cell stress was studied by using the cellular green fluorescence emission of specific probes (Invitrogen, USA) added to samples cultured at each treatment [44]. Cell viability was assessed with fluorescein diacetate (FDA), and 0.4 µL of a 0.09 µM working stock was added to 1 mL samples. FDA is a nonpolar, non-fluorescent stain, which diffuses freely into cells. Inside the cell, the FDA molecule is cleaved (hydrolysed) by nonspecific esterases to yield the fluorescent product fluorescein and two acetates. Accumulations of fluorescein are the result of intracellular esterase activity and thus indicate metabolic activity and therefore cell viability. ROS were assayed with carboxy-H2DFFDA, a cell-permeable fluorescent indicator that is non-fluorescent until oxidation by ROS occurs within the cell. H2DFFDA detects intracellular ROS species, and despite its lack of specificity, it has been proven very useful and reliable for assessing the overall oxidative stress being oxidized by any possible radical with oxidative activity [44]. ROS detection was performed after 15 µL of a 2 mM working stock of carboxy-H2DFFDA were added to 1 mL samples. Samples were incubated at 16 °C in darkness for 120 min under gentle shaking. Fluorescence was measured using an Accuri™ C6 flow cytometer (BD Biosciences, USA). Counts were triggered using forward scatter (FSC) signals.
Substrate Dependent Kinetics of Inorganic C Fixation
A 14C-based method was used to estimate the substrate dependent kinetics of inorganic C fixation based on Tortell et al. [45]. These measurements were conducted through short-term incubations of 10 min over a range of external C concentrations in buffered seawater (20 mM Bicine, pH 8.0). Prior to the beginning of the experiments, inorganic C was removed from the assay buffer by purging 20 mL aliquots with CO2-free air for at least 3 h [46, 47]. 1.5 mL of phytoplankton aliquots in C-free buffer were dispensed into polypropylene microcentrifuge tubes and placed in a custom-made, temperature-controlled glass chamber (16 °C). The incubation tubes were illuminated from the side with 600 µmol photons m−2 s−1 provided by a fluorescent tube of daylight type Osram Sylvania standard truelight 18 W. To initiate measurements, various amounts of 6 mM H12CO3− (0.108 g HCO3− + 20 mL of CO2-free water + 30 µL NaOH 4 N) and H14CO3− (DHI, Denmark) (specific activity vial: 2.18 × 109 Bq · mmol−1; final specific activity: 0.055 × 109 Bq · mmol−1; 2 mL stock of HCO3− cold + 0.2 mL ampoule of 14C (total 2.2 mL)) were added to each tube. The 14C/12C additions were adjusted to yield a final concentration of total inorganic carbon ranging from ≈ 50 to 4,000 µM, with a final specific activity of 0.055 × 109 Bq mmol−1. After 10 min of incubation, 500 µL of each tube were rapidly transferred into 500 µL of 6 N HCl in 20 mL scintillation vials and vortexed. Vials were then placed on a shaker table to degas evolved 14CO2 for at least 12 h. After this time, 14C activity of the samples was measured after adding 10 mL of scintillation cocktail (Ultima Gold, Perkin Elmer, USA) using a liquid scintillation counter (Packard Tri Carb Liquid Scintillation Analyser, Model 1900 A, Perkin Elmer, USA) with automatic quench correction. Background activity levels in cell-free blanks were subtracted from all samples.
Kinetic parameters Vmax and Km were derived from the 14C data using nonlinear, least-squares regression of the hyperbolic Michaelis–Menten equation (Eq. 2):
$$V={V}_{\mathrm{max}}*S/(S+{K}_{\mathrm{m}})$$
(2)
where V is the rate of C fixation at any given external C concentrations (S), and Vmax is the maximal rate of C fixation [note that maximal C fixation rates obtained from this analysis are not directly comparable to steady-state C uptake rates measured in traditional (12–24 h) 14C-incubation experiments. The 10-min rates reflect the total cellular capacity for C fixation, while longer-term rates include a significant contribution of respiration and organic C release, and cell death]. Km was the concentration of C for half Vmax. Carbon fixation rates were normalized by previously obtained cell counts.
Elemental Composition
Total particulate carbon (TPC) was measured using a C:H:N elemental analyser (Perkin-Elmer 2400 CHN). Twenty-five millilitres of each culture were gently filtered through pre-combusted (4.5 h, 500 °C) GF/F filters (Whatman) and dried at 60 °C for 24 h. For the determination of POC, the protocol was the same as for TPC, except those filters were fumed with saturated HCl overnight before analysis. PIC was assessed as the difference between TPC and POC.
Stable δ13C Isotopic Determination
The value of δ13C is used as a proxy of HCO3− versus CO2 only used by an aquatic primary producer, the former requiring a carbon concentrating mechanism (CCM). Typically, a value below (more negative than) − 30‰ indicates an inactive or absent CCM. However, this reference value should be taken cautiously, since it can be influenced by the specific δ13C value of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) for CO2 fixation in a given species. The abundance of 13C relative to 12C in E. huxleyi samples was determined by mass spectrometry using a DELTA V Advantage (Thermo Electron Corporation, USA) Isotope Ratio Mass Spectrometer (IRMS) connected to a Flash EA 1112 CNH analyser. δ13C isotopic discrimination in the microalgae samples (δ13Csample) was expressed in the unit notation as deviations from the 13C/12C ratio of the Pee-Dee Belemnite CaCO3 (PDB, which is the same as VPDB) calculated according to (Eq. 3):
$${\updelta }^{13}\mathrm{C}\left(\mathrm{\permil }\right)=[({\updelta }^{13}\mathrm{C}/{\updelta }^{12}\mathrm{C}{)}_{\mathrm{sample}}/({\updelta }^{13}\mathrm{C}/{\updelta }^{12}\mathrm{C}{)}_{\mathrm{PDB}}-1]\bullet {10}^{3}$$
(3)
To determine the isotopic composition of dissolved inorganic carbon (δ13CDIC), 25 mL from each cylinder were filtered (Whatman GF/F). Measurements of δ13CDIC were performed with the same IRMS mentioned above connected to a GasBench II (Thermo Electron Corporation) system. The δ13Csample was corrected by δ13CDIC values from the medium, previously tested in a TOC-L analyser.
pH Drift
A pH drift experiment was carried out to determine if E. huxleyi can use HCO3− as a source of inorganic carbon. The ability of algae to raise the pH of the medium to more than 9.0 is considered as evidence of their ability to use HCO3−. Samples were placed in 100 mL glass bottles filled (without leaving a head space) with 0.2 µL filtered seawater enriched with f/2 nutrients, and tightly sealed to avoid gas exchange. To obtain a complete homogenization of the medium, a magnetic bar was placed into each bottle and continuous stirring was provided by a magnetic stirrer. Samples were exposed to continuous illumination provided by white fluorescent tubes at saturating light. The pH was recorded by introducing a glass electrode through the lid of the glass bottle each 4–5 h until a stable reading was reached. Measurements were carried out until no further increase of the pH was detected.
Morphometric and Data Analysis of Coccoliths and Coccospheres
Size and morphological features of the cells were analysed using scanning electron microscopy (SEM). Samples of the different treatments (250 µL, 500 µL, and 1000 µL, depending on cell abundance) were filtered using Millipore Isopore™ hydrophilic polycarbonate membranes (RTTO01300) of 13 mm in diameter, and a pore size of 0.8 µm, using a vacuum pump under low pressure (< 200 mbar). Filters were rinsed with buffered distilled water to remove salt and then air dried overnight, mounted on aluminium SEM stubs, sputter coated with gold/palladium, and subsequently examined using a Zeiss EVO MA10 SEM.
The coccoliths were visually classified according to four morphological categories to estimate their degree of malformation [48, 49] (Fig. 6). The first category corresponds to normal coccoliths with all segments connected and forming an oval ring. The next three categories represent stages with increasing malformation signs characterized by a reduced symmetry, an altered shape of some of the elements, and reduced distal shield elements. Specifically, the second category corresponds to slightly malformed coccoliths, with less than 5 T-segments not well connected to others. The third category corresponds to malformed coccoliths where more than 5 T-segments are disconnected or not entirely formed. The fourth corresponds to fragmented coccoliths, in which parts of the coccoliths are missing. Category 4 is considered as severe malformation. The damaged coccoliths were measured only if their “reference points” could be unequivocally determined. Approximately 30 coccospheres and 30 coccoliths of each treatment were analysed (i.e., HC-LP, LC-HP, and LC-LP). The mean values of each parameter were considered constant when there were more than 20 coccospheres and coccoliths measurable per sample [50]. As for the coccoliths, all the morphometric measurements were performed on the distal shield of flat lying E. huxleyi placoliths (see Supplementary Fig. S2). Measurements included the length of the distal shield (DL), the width of the distal shield (DW), the length of the central area (CAL), and the width of the central area (CAW). CAL and CAW could not be determined in cases where the coccolith was lying upside-down on the filter. In addition, the number of segments or elements that form the distal shield were recorded. The surface area of the distal shield (DSA) was estimated with the values of DL and DW [51] (Eq. 4):
$$DSA=\pi \cdot \frac{DL\cdot DW}{4}$$
(4)
The outer shield length (OSL) was calculated assuming an elliptical shape of coccolith, such as (Eq. 5):
$$OSL=\frac{DL-CAL+DW-CAW}{4}$$
(5)
In addition, the calculation of the surface area of central shield (CSA) is proposed taking the values of CAL and CAW, such as (Eq. 6):
$$CSA=\pi \cdot \frac{CAL\cdot CAW}{4}$$
(6)
The width of the tube (protococcolith ring) (TW) varies between coccoliths of E. huxleyi, from slightly calcified coccoliths in which the central area is wide and the tube is narrow to very calcified coccoliths in which the central area is almost closed. To obtain a size independent parameter to measure this degree of calcification variation, we used relative tube width (TWrelative) (Eq. 7). This parameter is used here as a calcification index. This ratio is dimensionless and should be size-independent.
$${TW}_{\mathrm{relative}}=\frac{2\bullet TW}{DW}$$
(7)
Coccoliths mass (m) has also been used as an indicator of the impact of OA on coccolithophores [52, 53] as (Eq. 8):
$$m=2.7\bullet {k}_{\mathrm{s}}\bullet {DL}^{3}$$
(8)
where ks is a shape dependant constant, Ks = 0.07 TWrelative, and DL distal shield length.
The roundness of the distal shield (DR) (Eq. 9) and the roundness of the central area (CAR) (Eq. 10) were calculated, from the ratio of their width and length measurements [54] as:
$$DR=DW/DL\bullet 100$$
(9)
$$CAR=CAW/CAL\bullet 100$$
(10)
As for the coccospheres, two measurements were made, one of them corresponding to the greater length L and another to the shorter length W.
Measurements were taken from SEM micrographs which were processed and analysed using the software Fiji-ImageJ 1.49v software [55, 56] (National Institute of Health, USA).
Statistical Analyses
Statistical significance of treatments was analysed by performing split-plot ANOVAs (SPANOVAs, or mixed-model ANOVAs) followed by post hoc Sidak or Tukey and Bonferroni tests, respectively (considering p < 0.05 as significant). When appropriate, data were specifically tested for significant differences (p < 0.05) induced by the treatments by using 1-way ANOVAs and/or Student’s t-tests, as well as Pearson’s product-moment correlations. All analyses were performed using the general linear model (GLM) procedure. Data were previously checked for normality (by Shapiro-Wilks’ test), homoscedasticity (by Cochran’s and Levene’s tests), and sphericity (by Mauchly’s and/or Bartlett’s tests). Variables met all criteria mentioned above. Statistical analyses were performed using the software SPSS v.22 (IBM statistics) and R-studio.