An Abrupt CO2-Mediated Decrease In

Limnetica, 37(2): 267-281 (2018). DOI: 10.23818/limn.37.22 © Asociación Ibérica de Limnología, Madrid. Spain. ISSN: 0213-8409

An abrupt CO2-mediated decrease in pH affects growth rates, cellular features and the interspecific interaction of Scenedesmus (Acutodesmus) obliquus and Cryptomonas pyrenoidifera

Andréa Galotti*, Francisco Jiménez-Gómez and Gema Parra

Department of Animal Biology, Plant Biology and Ecology and Centre of Advanced Studies in Earth Sciences, University of Jaén, 23071 Spain.

* Corresponding author: [email protected]

Received: 11/05/17 Accepted: 18/04/18

ABSTRACT An abrupt CO2-mediated decrease in pH affects growth rates, cellular features and the interspecific interaction of Scenedesmus (Acutodesmus) obliquus and Cryptomonas pyrenoidifera The technologies of anthropogenic CO2 mitigation, such as carbon capture and sequestration, may pose an environmental threat to aquatic systems. In a scenario of CO2 leakage from a carbon capture and sequestration process, very low-pH values might be reached and could remain over time. The main objective of this study was to detect how an abrupt lowering of pH would affect the microalgae Scenedesmus (Acutodesmus) obliquus and Cryptomonas pyrenoidifera at physiological, morphological and population levels, and also see how these effects could lead to ecological consequences. Monospecific and mixed culture experiments were run according to this purpose over 14 days and at a pH of 6.5, controlled by CO2 injection. An increased CO2 concentration significantly enhanced the growth rate of both species and especially affected the cell size of C. pyrenoidifera in the monoculture. The total biovolume of C. pyrenoidifera was higher than the total biovolume of S. obliquus in the control treatment, although neither of the two species were dominant in the culture experiments. Granularity responded in different ways for the species studied, being statistically different within subjects in monospecific and mixed culture experiments. Only chlorophyll and granularity have been significantly correlated in the low pH of C. pyrenoidifera monoculture. Due to its ecological relevance, the decreased colony formation ability of S. obliquus under a high CO2 concentration is highlighted. Key words: CO2 bubbling, Colony Formation, Cryptomonas pyrenoidifera, Flow Cytometry, Scenedesmus (Acutodesmus) obliquus

RESUMEN Un abrupto descenso de pH mediado por inyección de CO2 afecta a la tasa de crecimiento, a las características celulares y la interacción interespecífica de Scenedesmus (Acutodesmus) obliquus y Cryptomonas pyrenoidifera Las tecnologías de mitigación del CO2 antropogénico, como la captura y secuestro de carbono, pueden constituir una amenaza ambiental para los sistemas acuáticos. En un escenario de fuga de CO2 durante el proceso de captura y secuestro de este gas, se pueden alcanzar valores muy bajos de pH. El objetivo principal de este estudio fue estudiar cómo una bajada abrupta de pH afecta a nivel fisiológico, morfológico y poblacional a las microalgas Scenedesmus (Acutodesmus) obliquus y Cryptomonas pyrenoidifera, y, cómo estos efectos podrían tener consecuencias ecológicas. De acuerdo con este propósito, se llevaron a cabo experimentos con cultivos monoespecíficos y también cultivo de mezcla de ambas especies durante 14 días a un pH 6.5, mediante inyección de CO2. La exposición a altas concentraciones de CO2 aumentó significantemente la tasa de crecimiento en ambas especies y alteró especialmente el tamaño celular de C. pyrenoidifera en el monocultivo. El biovolume total fue estadísticamente diferente entre individuos de la misma especie expuestos a condiciones control y de pH 6.5. El biovolumen total de C. pyrenoidifera fue mayor que el de S. obliquus en el control, aunque ninguna de las dos especies fue dominante (en biovolumen) en el experimento de mezcla de especies. La granularidad respondió de manera distinta en las especies estudiadas, aunque en ambos casos mostraron diferencias entre tratamientos. Solamente la clorofila y la granularidad han estado significantemente correlacionadas en el tratamiento a bajo pH del monocultivo de C. pyrenoidifera. Debido a

su relevancia ecológica, es necesario destacar la disminución de la capacidad de formación de colonias de S. obliquus bajo condiciones de altas concentraciones de CO2. Palabras clave: Burbujeo CO2, Citometría de flujo, Cryptomonas pyrenoidifera, Formación de colonias, Scenedesmus (Acutodesmus) obliquus An abrupt CO2-mediated decrease in pH affects growth rates, cellular features and the interspecific interaction of Scenedesmus (Acutodesmus) obliquus and Cryptomonas pyrenoidifera

Andréa Galotti*, Francisco Jiménez-Gómez and Gema Parra

Department of Animal Biology, Plant Biology and Ecology and Centre of Advanced Studies in Earth Sciences, University of Jaén, 23071 Spain.

* Corresponding author: [email protected]

Received: 11/05/17 Accepted: 18/04/18

268 Galotti et al. su relevancia ecológica, es necesario destacar la disminución de la capacidad de formación de colonias de S. obliquus bajo Growth rate, Cell size, Granularity, Single/colony For this purpose, monospecific culture experi- condiciones de altas concentraciones de CO2. cell percentage and Biovolume. The cosmopolitan ments and a competition experiment were carried Palabras clave: Burbujeo CO2, Citometría de flujo, Cryptomonas pyrenoidifera, Formación de colonias, Scenedesmus freshwater microalga, Scenedesmus (Acutodesmus) out with both species for 14 days. The competi- (Acutodesmus) obliquus obliquus (Turpin) Kützing (single cells and tion experiment tries to clarify the interspecific coenobia – S. obliquus from here) and Crypto- interaction under CO2-mediated low pH. Species monas pyrenoidifera Geitler (single flagellated differences in responses could lead to a shift in INTRODUCTION Elevated CO2 concentration will result in a cells – C. pyrenoidifera from here) were used as the community with a great ecological concern. decrease in pH which might affect intracellular testing organisms due to the fact they are easy to Besides conventional light-inverted microscopy, It is expected that atmospheric carbon dioxide pH, altering many physiological and structural handle and culture under experimental conditions flow cytometry is proposed as a potential tool for (CO2) will double over the next one hundred processes (Segovia et al., 2018). Moreover, it is and to the existence of previous information. Both phytoplankton optical properties analysis in this years (Solomon et al., 2007). Carbon capture and well documented that cells grown under high species are widely distributed around the world context of carbon capture and sequestration geological storage (CCS) technologies are among CO2 conditions have a less developed or an (Cepák et al., 2007; Guiry & Guiry, 2018) and environmental risk assessment, which allows rapid the carbon mitigation strategies being currently undetectable pyrenoid (Miyachi et al., 1986). sometimes in coexistence, e.g. in a reservoir in analyses of the growth rate and cell features and had discussed (IPCC, 2015; Szalaj et al., 2017). Many Besides, Morita et al. (1998) demonstrated Turkey (Sevindik, 2010) and in a subtropical lake previously been used in the monitoring of the physi- studies have been carried out regarding the risk of with non-pyrenoids algae that the presence of in China (Chen et al., 2008). ological state of microalgal cells (Cid et al., 1996). leakage from the delivery and geological storage pyrenoids or the accumulation of RuBisCO in of large volumes of CO2 (e.g. Basallote et al., chloroplasts is not always essential for the carbon 2012), where pH might be even lower than 4 pH concentration mechanism (CCM). Therefore, units (IPCC, 2005) and cause potential environ- physiological and fine structure properties of the mental consequences. Such acidification might cells could undergo changes in high CO2 concen- affect populations that inhabit the environment tration. These changes in the inner features of the around the pipe where the leakage happens. Envi- cell can be detectable by flow cytometry such as ronmental impacts on aquifers and in their micro- granularity (Shapiro, 1995). On the other hand, bial community have been described (Morozova the reduction of colony formation ability in S. et al., 2010), as well as hydro-chemical changes obliquus has been described under acidification associated with the upward migration of stored adjusted with HCl- (Yang et al., 2016) and CO2 through faults, fractures and poorly sealed or CO2-enhanced concentration (Huang et al., abandoned wells into shallow drinking water 2017). This was also reported in the species aquifers (Yang et al., 2014). The aquifer Botryococcus braunii when the size of the colony recharge-discharge is a relevant part of the suffers changes under high CO2 concentration hydrology of aquatic systems and CO2 leakages (Ge et al., 2011). This inducible defence plays an could have consequences on them. important role in preventing two important Microalgae are responsible for most primary ecological challenges faced by the microalgae, production in aquatic ecosystems and fuel energy sedimentation and grazing rates (Verschoor et al., transfer for the rest of the food web of these 2004; Stap et al., 2006). Thus, studies to reveal ecosystems, including the microbial loop (Cairns the effect of high CO2 concentration on the et al., 1992;). The pH of water affects algal colony formation of microalgae are necessary. growth and survival (e.g. Gensemer et al., 2018). The working hypothesis is that a low pH, The first studies carried out in the last third of the caused by a high concentration of CO2 in the twentieth century suggested that the high CO2 medium will affect microalgae at three levels: concentration in the medium would reduce the physiological, morphological and population efficiency in CO2 fixation by microalgae (Wer- levels (e.g. colony feature changes), that could dan et al., 1975; Coleman & Colman, 1981). lead to ecological consequences. In order to check Primary production is expected to be stimulated this hypothesis, we developed laboratory-based in the short-term by the CO2 increase (Hein & manipulative experiments, which aimed to Sand-Jensen, 1997), so that one of the most disclose the effects of low pH caused by an injec- prominent concerns about increased CO2 is that it tion of CO2 on the growth rates and cellular would induce uncontrollable primary production features in two microalgae used as experimental (see the review by Riebesell & Tortell, 2011). models. The selected endpoints were Cell density,

Limnetica, 37(2): 267-281 (2018)

MATERIAL AND METHODS test and control vessels. Five replicated 500 ml were Flow-CheckTM and Flow-SetTM, both from Significant differences between treatments vessels for low pH (pH 6.5) and four for control Beckman Coulter, Inc.; Yellow-Green were tested by means of a repeated measures Cultured species were set with an initial experimental density of Fluospheres from Molecular Probes and BD ANOVA and a DMS post-hoc test when suitable. approximately 2.0 x105 cells/ml S. obliquus and CalibriteTM. The equipment utilised was a Requirements for the analysis (normality and S. obliquus (from Chemical Engineering Laborato- 2.0 x104 cells/ml C. pyrenoidifera in the mono- BD-LSR Fortessa, with slow flow (12 μl/minute) homoscedasticity) of variance were tested in all ry, University of Jaén, Spain) and C. pyrenoidifera culture, and 1.6 x105 cells/ml S. obliquus and 1.5 for 180 seconds while recording optical properties cases. Significance levels were tested at the p = (from Water Research Institute, University of x104 cells/ml C. pyrenoidifera in the mixed from each cell, focusing on forward scattering 0.05 level. Both between-subject and within-sub- Granada, Spain) were grown in 3N-BBM+V culture. The difference between species’ initial (FSC-channel related to cell size), side scattering ject were tested for significance. In addition, linear medium (recipe from CCAP, Scotland; pH concentrations were chosen given their individual (SSC-channel related to size and granularity) and regression was tested for significance when corrected to 8.3-8.5 with NaOH before sterilisa- biovolumes (C. pyrenoidifera is bigger than S. red fluorescence (PerCP-Cy5-5-channel related to required. Means are presented ± standard deviation tion by autoclaving). Cultures were maintained in obliquus), which calculations are explained chlorophyll fluorescence). Cell densities were (SD), where n = number of vessels for each experi- a temperature-humidity-light controlled chamber below, so that the total biovolume for both calculated from the nominal flow rate of the ment, as specified above unless otherwise stated. at 20 ºC, supplied with a 12:12 h light cycle and species will be similar in each vessel. In addition, cytometer and the acquisition time. Data were there was no limitation of growth by light satura- the initial densities are lower for each species in acquired and analysed obtaining the geometric RESULTS tion (Sorokin & Krauss, 1958) with an irradiance the mixed culture in order to prevent saturation. mean values (Geo-Mean; Campbell 1995) of each of about 200 μmol photons m-2 · s-1 (Flöder et al., The initial individual biovolume was calculat- parameter from the different clouds. Abundance is In control treatments, the pH monitored by the 2006; Gris et al., 2014). ed in order to find out the space occupied by each calculated according to the number of points system showed that the pH rose to values of species and so to decide the initial density used located on the species cloud, the time of sample 8.5-9.0 as a consequence of the algal metabolism. CO2 Laboratory-Based Manipulative Experi- for each species in the competition experiment, so acquisition and its flow velocity acquisition. Treatment vessels with low-pH were maintained ments saturation by one of them would be prevented. Single and colony formation has been measured at pH 6.5 (±0.02) with controlled injections of Several cells of each species were measured from using the dot clouds from flow cytometry. In CO2 during the experimental period, and conse- The experiments were carried out using a system photographs taken with a digital camera (Leica addition, the approximate biovolume of S. obliquus quently no overlap occurred between treatments. of CO2 bubbling into the water described in detail EC3) and then analysed using the software Image events in the monoculture was calculated from the Results are organized in both tables and by Basallote et al. (2012). All the experimental J (U. S. National Institutes of Health, Bethesda). antilog transformation of the FSC-H channel. The figures. Table S1 (S. obliquus monoculture), Table vessels contained a pH sensor connected to the Biovolumes were calculated following the equa- actual value of FSC-H was multiplied by the S2 (C. pyrenoidifera monoculture), Table S3 (S. Aqua Medic AT Control System, which included tions proposed by Hillebrand et al. (1999) for a number of events in their respective channel, and obliquus competition experiment) and Table S4 the CO2 bubbling pump as well as the air pump in prolate spheroid shape. Individual biovolume was that value was designated as an approximate (C. pyrenoidifera competition experiment) can be order to decrease the rate of cell sedimentation. established according to the mean size of each biovolume. In order to visualize the existence of consulted on the supplementary information avail- Before use, pH electrodes were calibrated, and species, as 104.7 μm3 for S. obliquus and 1014.3 internal biases in the dot clouds, the accumulated able at http://www.limnetica.net/en/limnetica. the values obtained throughout the tests were μm3 for C. pyrenoidifera. Following the equation approximate biovolume was calculated. These tables present results of the proposed regularly verified by a portable pH-meter (Crison to estimate the biomass carbon content by Rocha Growth rate (GR) was also calculated after endpoints (cell density, growth rate, cell size, gran- GLP 22). CO2 pumps were controlled by a & Duncan (1985), initially S. obliquus has a flow cytometric results following GR=[lN(Nf)-lN ularity, chlorophyll content, single/colony cell solenoid valve that stops the CO2 input when the content carbon of 15.98 whereas C. pyrenoidifera (N0)]/(lN2 (t-t0)), where GR is growth rate · percentage and total biovolume) plus their SD for system detected that the one-vessel pH had has 173.82 pg C μm-3. day-1, t0 and t are the initial and final days in the each day of analysis (day 0, 1, 2, 7, 10 and 14) reached the established level. A computer In the competition experiment, initial densi- study period, respectively, and N0 and Nf are the under control and low-pH treatments. connected to the AT control system permitted ties were calculated following the total biovol- cell density (cells/ml) at the initial and final days modification of the pH values (for more details, ume of each species (total biovolume is calculat- of study, respectively. S. obliquus monoculture please see Fig. 1 on Bautista-Chamizo et al., ed by individual biovolume multiplied by the Samples were also taken for microscopy 2016: 957). number of cells), so 50 % of the total biovolume analysis, preserved in Lugol’s solution (2 %) for Algal cell density of S. obliquus increased during Three different experiments were set up, contained in the vessels were occupied by each the initial individual biovolume measurements (S. the experiment under low-pH and control treatment being (i) S. obliquus monoculture; (ii) C. pyre- species. This calculation was also used to assess obliquus and C. pyrenoidifera) and the analysis of (See Table S1). Significant differences were noidifera monoculture; and (iii) S. obliquus plus species displacement at the end of the experi- the percentage of single cells and colonies (just observed between subjects (low-pH and control C. pyrenoidifera mixed culture (competition ments. Flow cytometric analyses were conducted for S. obliquus). For the colony analysis, at least treatments; See Table 1). Cell density in low-pH experiment from here). All of them were carried immediately after sampling in order to avoid loss 200 cells were counted randomly on a Sedgwick treatment was higher than the control throughout out over a 14-day period. Samples from each of fluorescence and damage in living cells. Flow Rafter Chamber under a Leitz DMIL inverted the experimental period, starting from 22.7 % vessel were monitored on Day 0 (inoculum day), cytometry settings were adapted to the sizes and microscope (40x magnification). All the colonies higher on day 1 up to 240.2 % on day 7, when the 1, 2, 7, 10 and 14. fluorescence emission of S. obliquus and C. pyre- found were counted as “one colony” inde- maximum difference was observed. The experi- The same conditions of stock culture as men- noidifera. Four different calibrating beads were pendently of whether the colony (coenobia) was ment finished with cell density in low-pH treatment tioned above (Cultured species) were kept for the used following Galotti et al. (2006). These beads formed by two, four or eight cells. being higher than the control at 27.6 % on day 14.

S. obliquus growth rate (GR) in the monocul- (PerCP-Cy5-5-H geometric mean = red fluores- ture was significantly higher in the low-pH than in cence) and granularity (SSC-H geometric means) the control treatment (See Table 1). The biggest (Fig. 1b). The correlations between cell size and difference between GR in low-pH and control granularity in S. obliquus monoculture were simi- treatments was seen on day 2 (See Table S1). lar for both the control and the low-pH treatments The biggest differences in cell size (FSC-H (F1,19= 166.368; p= 0.000; r2= 0.8986 for the Geo Mean value) between treatment and control in control and F1,25= 247.314; p= 0.000; r2= 0.8947 monoculture were found on day 1 (See Table 1). for the low-pH treatment; Fig. 1a). There was a However, no significant differences were found correlation between chlorophyll and granularity after 7 days of the experiment (See Table 1). in the control treatment (F1,18= 61.575; p= 0.000; The oscillatory fashion of granularity was similar r2= 0.7789), while there was not under low-pH between the control and the low-pH treatments (p> 0.05; r2= 0.0361) (Fig. 1b). during the experimental period. Values of granulari- The percentage of colonies in relation to ty were only higher in the control than in the low-pH single cells of S. obliquus decreased in the treatment on days 1 and 2 (See Table S1). low-pH treatments of monoculture (See Table In order to assess that granularity is related to S1) and was significantly different from control changes in inner features rather than to cell size (See Table 1), indicating lower colony formation only, two correlations were performed. The first ability under 6.5 pH. one between cell size (FSC-H geometric means) To confirm the low colony formation in and granularity (SSC-H geometric means) (Fig. low-pH treatment, flow cytometry cytograms 1a), and the second between chlorophyll were used. Figure 2 illustrates the response in

accumulated approximated biovolume (µm3) in fashion in the control and low-pH treatments, each treatment, considering the number of cells in always in the contrary manner between treatments each FSC channel multiplied by the calibrated (See Table S2). Granularity was correlated with size of each channel. As in the accumulative cell size and chlorophyll content (Fig. 1c and d, probability curves, this representation allows the respectively). A significant negative correlation detection of deviation from normal distributions between granularity and red fluorescence (chloro- in dense continuous dot clusters. The control phyll content) was only observed in the low-pH curve showed two inflection points that corre- treatment (F1,16= 22.323; p= 0.000; r2= 0.6002). sponded to two subpopulations (singles and colony) within the cluster of this species. How- Competition experiment ever, in the low-pH treatment the accumulated curve showed a single inflection point, at the As expected, the cell density of S. obliquus and level of the smaller subpopulation. C. pyrenoidifera was significantly different between species throughout the experimental C. pyrenoidifera monoculture period, since the initial cell density was already different in order to accomplish similarities in C. pyrenoidifera had its growth stimulated in the total biovolume between species. However, only low-pH. Cell density was significantly lower in S. obliquus cell density was statistically different control (p> 0.05; See Table 1) on day 14. By the between treatments, low-pH and control (See end of the experiment, the cell density in the Table 1). S. obliquus cell density was lower low-pH treatment recovery was 28.5 % higher under the low-pH treatment of the competition than in the control on day 14 (Table S2). experiment than in monoculture. Meanwhile, C. The C. pyrenoidifera growth rate (d-1) was not pyrenoidifera cell density under low-pH was significantly different between treatments (See similar in both experiments. Table 1), and only slight differences were detected At the end of the competition experiment, S. during the initial days (day 1 and 2; See Table S2). obliquus growth rate was higher under low-pH Significant differences were seen on days 7, than control but lower in both cases than those in 10 and 14 when the cell size in the low-pH treat- the monoculture. Differences were significant ment was bigger than in the control (see Table S2; both between and within subjects (See Table 1). and Table 1 for significance levels). The C. pyrenoidifera growth rate was higher C. pyrenoidifera granularity (SSC-H geomet- under low-pH than the control at the end of the ric mean) responded in a somewhat oscillatory competition experiment, being even higher than the growth rates in both treatments in the mono- mentation, ending larger than on the initial day. In culture (See Table S2 and S4). Responses were addition, it was larger in the control than in the significantly different among species and treat- low-pH treatment (See Table S3 and Table 1). ments on day 2, day 10 and day 14 (p< 0.05). In the competition experiment, the response of In order to compare the growth rates between C. pyrenoidifera cell size observed between treat- monoculture and the competition experiment, ments was similar to monoculture, increasing the growth rate variation percentage was calcu- both in the low-pH and control but higher in the lated (see Fig. 3). The highest percentage of low-pH treatment as well as in the monoculture variation was observed on the first day of exper- (See Table S4). imentation for both species, S. obliquus (Fig. 3a) S. obliquus granularity (SSC-H values) has and C. pyrenoidifera (Fig. 3b). From the second shown the same pattern during the experiment under day, the variation in S. obliquus remained nega- the low-pH and control treatments (See Table S3), tive in the control and the low-pH treatments. while C. pyrenoidifera granularity was higher in the The variation percentage in C. pyrenoidifera low-pH treatment than in the control treatment, remained positive from day 7. especially on days 7 and 10 (See Table S4). The The cell size of S. obliquus responded in an difference throughout the period of the experiment oscillatory fashion during the period of experi- was significant for both species (See Table 1).

S. obliquus granularity was significantly corre- obliquus in our study. On the one hand, the experiment, differences in cell size were also Franqueira et al. (1999) found an increase in lated with cell size in control (F1,11= 178.939; response of S. obliquus as well as C. pyrenoidifera significant. The ability of Scenedesmus to change multivesicular bodies in the cytoplasm as a conse- p= 0.000; r2= 0.9471) (Fig 4a). However, there under low pH was different in the monospecific its cell size is well known (Trainor, 1998). Such quence of pesticide exposure. In this sense, Frank- was no correlation between S. obliquus granular- and competition experiments. Specifically, in the changes in cell features could have greater conse- lin et al. (2001) had already suggested that the ity and chlorophyll content in any of the treat- presence of C. pyrenoidifera, S. obliquus reduced quences in higher hierarchical levels. Herbivore light-scattering properties of two algal species ments (Fig. 4b). In addition, no correlation was its growth rate, and it was even lower than those grazing would be promoted if cell size or induced (Chlorella sp. and Phaeodactylum tricornutum) observed between C. pyrenoidifera granularity found by other authors (Yang & Gao, 2003) under colony formation are impaired. Besides, the abili- could be an alternative as chronic test endpoints. and cell size (Fig. 4c), but positive correlation was similar CO2 conditions. On the other hand, ty of S. obliquus to form colonies has been deplet- The changes might be correlated with the com- observed between C. pyrenoidifera granularity and Low‐Décarie et al. (2011) found that S. obliquus ed. The percentage of single-cells and colonies of plexity of the cytoplasm (e.g. Reader et al., chlorophyll content in the low-pH treatment displaced the cyanobacteria under high CO2 and S. obliquus were altered by low pH. Scenedesmus 1993), hence with photosynthetic structures such (F1,27= 64.244; p= 0.000; r2= 0.7119) (Fig. 4d). within a mixed culture. usually forms colonies of four or even eight cells as chloroplasts. In this context, high levels of CO2 In the competition experiment, the percentage It is known that high CO2 concentration will (coenobia) as a strategy against grazing (see enhance the production of drops of lipids (initially of colonies in relation to single cells of S. cause unprecedented changes, the consequences Lürling, 2003 for a review; Wu et al., 2013). “This set to pH 6.5 in Cheng et al., 2013) driven by obliquus decreased in the low-pH treatment (See of which might be difficult to predict, since some ability of a single genotype to produce one or increasing the flux of CO2 to acetyl-CoA (acetyl Table S3) and were statistically different than organisms may respond positively while many more alternative forms of morphology in response coenzyme A) in microalgal chloroplasts (called control (See Table 1). This response was similar others are likely to be at a disadvantage to environmental conditions is termed phenotypic “high doses” of CO2 aeration 10 % (v/v) in Sun et to the monoculture experiment; however, the (Assunção et al., 2017; Choix et al., 2018). For plasticity” (West-Eberhard, 1989) and, as afore- al., 2016). Although granularity was a suitable percentage of colonies in the control of competi- example, the CCM differ among microalgae mentioned, operates such a protection against endpoint to C. pyrenoidifera, as a measurement of tion experiment was much higher than in the species (Rost et al., 2003; Ainsworth & Long, grazing for zooplankters (Wu et al., 2013). Yang internal cellular changes, it was not to S. obliquus. control of the monoculture. 2005), especially concerning their efficacy for et al. (2016) found, in a study performed over 7 The oscillatory fashion in the internal cellular Tables S3 and S4 also show the total biovol- growing (Fu et al., 2008; Kardol et al., 2010), e.g. days, that S. obliquus colony formation induction granularity of C. pyrenoidifera might be also ume of both species in the competitive experi- spending less energy on CCM that can be invest- is depleted under the pH from 5 to 9 adjusted with explained by the presence of different pigments in ment. The total biovolume of both species under ed in growth processes (Giordano et al., 2005). HCl. In this sense, CO2-induced low pH likewise the Cryptomonads group, such as carotenoids the low-pH treatment was significantly different High CO2 might also result in community shifts altered such functioning in the S. obliquus cells, (Chi c2) and cryptomonad phycoerythrin on days 7 and 10 but was similar at the end of the with deep ecological consequences for the whole somehow preventing colony formation and addi- (Cr-PE565; Mimuro et al., 1998) which could be experiment (Table 1). The total C. pyrenoidifera system (Hutchins et al., 2009). One of the most tionally, making the cells more exposed to the altered by the high CO2 concentration medium biovolume percentage in the low-pH treatment concerning effects in the early stages of a high medium. This feature was easily detectable by (for further information, see Rmiki et al., 1999). decreased throughout the last three days in com- CO2 concentration in water bodies is eutrophica- flow cytometry also, and the decrease in colony Apparently, following the biovolume percent- parison to its values in the control treatment tion, being responsible for an ecological conse- formation remained throughout the 14 days of the age, neither of the species would displace the (Table S4). The highest total biovolume of S. quence over the long term. The results presented experimental period, both in monoculture and in other. Although the results of our competition obliquus was observed on the last day (day 14, here demonstrate that S. obliquus reduced its competition experiment. The reduction of colony experiment have not provided sufficient evidence Table S3). growth capacities at the beginning of the compe- formation under low-pH might be related to mem- to state that the displacement of species will The slopes of biovolume percentage under tition culture under low pH; this could lead to brane transport processes (i.e. depolarization of happen, if the tendency to increase the total low-pH and control were calculated in order to changes in the community structure compared the membrane potential in S. obliquus has been biovolume percentage of S. obliquus would display the trend of C. pyrenoidifera displace- with the control condition. According to the observed; unpublished data) and metabolic func- remain for a longer term, both eutrophication and ment performed by S. obliquus. The slope values resource competition theory, two species that are tions involved in algal cellular pH regulation displacement of cells might occur. from day 7 to day 14 were 2.4534 (r2 = 0.6028) limited by two or more resources can coexist (Jiang et al., 2012). The colony formation ability Amidst some limitations, the experiments and 0.7011 (r2 = 0.083) for low-pH and control, (Tilman, 1982); in a mixed culture at low-pH they was a suitable endpoint for S. obliquus as it shows could be used to check out the linkage between respectively. Slopes were significantly different would coexist in a somewhat oscillatory pattern a CO2 exposure effect with ecological relevance. the physiological responses (endpoints) proposed according to the statistical analysis (p< 0.001). (Verschoor et al., 2013). Although the process of Without this defence mechanism, these microal- and the response of microalgae species, in order biodiversity loss under conditions of ocean acidi- gae might suffer a higher grazing rate, which to ascertain any shifts in the community dynam- DISCUSSION fication is well understood (i.e. Booth et al., could lead to a higher growth rate of the zooplank- ics that would be attributable to changes in CO2 2018) this is not the case for other water bodies. ton, which in turn could lead to an imbalance in concentration, hence in pH. The studied Scenedesmus obliquus grew more efficiently in In the present study, the two species studied the trophic network. However, further research endpoints: cell density, growth rate, cell size, the low pH treatment, while C. pyrenoidifera changed their individual size in response to focused on how a CO2-mediated low pH alters granularity, single-cell/colonies percentage and reached similar densities between treatments. low-pH treatment. In the monoculture low-pH physiologically the colony formation mechanism total biovolume were suitable to show the effects Moheimani & Borowitzka (2011) found that the treatment, S. obliquus increased its cell size, should be conducted. of low pH on microalgae. For example, regarding microalga Pleurochrysis cearterae seemed to act whereas C. pyrenoidifera decreased its cell size Granularity, measured with flow cytometry, cell size, although the size of S. obliquus as a CO2 user, which might be the case of S. under the same conditions. And in the competition has previously been used as an endpoint, i.e. decreased, its granularity in low-pH treatment

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Eval- rapid technique in the present study being deter- Á. DELVALLS & I. RIBA. 2016. Simulating sured by flow cytometry. Cytometry, 25: 32-36. uating the effects of pH, hardness, and minant in providing quick and reliable results. CO2 leakages from CCS to determine Zn toxic- DOI: 10.1002/(SICI)1097-0320(19960901) dissolved organic carbon on the toxicity of In conclusion, the elevated CO2 concentration ity using the marine microalgae Pleurochrysis 25:1<32::AID-CYTO4>3.0.CO;2-G aluminum to freshwater aquatic organisms modified the growth dynamics and specific cell roscoffensis. Chemosphere, 144: 955-965. COLEMAN, J. R. & B. COLMAN. 1981. under circumneutral conditions. Environmen- features in both species as well as the interaction DOI: 10.1016/j.chemosphere.2015.09.041 Inorganic carbon accumulation and photosyn- tal Toxicology and Chemistry, 37: 49-60. between them. BOOTH, D. J., E. POLOCZANSKA, J. M. thesis in blue-green-alga as a function of DOI: 10.1002/etc.3920 DONELSON, J. G. 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Economía y Competividad, Junta de Andalucía, tion as a model for bio‐optical variability in the FRANQUEIRA, D., A. CID, E. TORRES, M. HEIN, M. & K. SAND-JENSEN. 1997. CO2 European Regional Development Fund) is grate- sea. Journal of Geophysical Research-Oceans, OROSA & C. HERRERO. 1999. A compari- increases oceanic primary production. Nature, fully acknowledged. Undoubtedly, our thanks are 100: 13237-13254. DOI: 10.1029/95JC00458 son of the relative sensitivity of structural 388:526-527. DOI: 10.1038/41457 also due to the editor of this Journal and to the two CEPÁK, V., P. PRIBYL, M. VÍTOVÁ & V. and functional cellular responses in the alga HILLEBRAND, H., C.D. DÜRSELEN, D. referees who reviewed the manuscript in detail. ZACHLEDER. 2007. The nucleocytosolic and Chlamydomonas eugametos exposed to the KIRSCHTEL, U. POLLINGHER & T. chloroplast cycle in the green chlorococcal herbicide paraquat. Archives of Environmetal ZOHARY. 1999. 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Genetic diversity of eukaryotic microor- temperature effects on sympatric Heterosigma enhances grazer‐induced morphological properties and plant production to rising CO2. ganisms in Lake Taihu, a large shallow subtrop- akashiwo and Prorocentrum minimum. Harm- defense in the freshwater green alga Scened- New Phytologist, 165: 351-372. DOI: ical lake in China. Microbial ecology, 56: ful Algae, 7: 76-90. DOI: 10.1016/j.hal.2007. esmus obliquus. Limnology and Oceanogra- 10.1111/j.1469-8137.2004.01224.x 572-583. DOI: 10.1007/s00248-008-9377-8 05.006 phy. DOI: 10.1002/lno.10715 ASSUNÇÃO, J., A. P. BATISTA, J. MANOEL, CHENG J., Y. HUANG, J. FENG, J. SUN, J. GALOTTI, A., F. JIMÉNEZ-GÓMEZ & F. HUTCHINS, D.A., M.R. MULHOLLAND & F. T. L. DA SILVA, P. MARQUES, A. REIS & ZHOU & K. CEN. 2013. Mutate Chlorella sp. GUERRERO. 2006. Estructura de tamaños de FU. 2009. Nutrient cycles and marine L. GOUVEIA. 2017. CO2 utilization in the by nuclear irradiation to fix high concentrations las comunidades microbianas en sistemas microbes in a CO2-enriched ocean. Oceanog-

Andréa Galotti*, Francisco Jiménez-Gómez and Gema Parra

Department of Animal Biology, Plant Biology and Ecology and Centre of Advanced Studies in Earth Sciences, University of Jaén, 23071 Spain.

* Corresponding author: [email protected]

Received: 11/05/17 Accepted: 18/04/18

CO2-mediated low pH affects microalgae 269 su relevancia ecológica, es necesario destacar la disminución de la capacidad de formación de colonias de S. obliquus bajo Growth rate, Cell size, Granularity, Single/colony For this purpose, monospecific culture experi- condiciones de altas concentraciones de CO2. cell percentage and Biovolume. The cosmopolitan ments and a competition experiment were carried Palabras clave: Burbujeo CO2, Citometría de flujo, Cryptomonas pyrenoidifera, Formación de colonias, Scenedesmus freshwater microalga, Scenedesmus (Acutodesmus) out with both species for 14 days. The competi- (Acutodesmus) obliquus obliquus (Turpin) Kützing (single cells and tion experiment tries to clarify the interspecific coenobia – S. obliquus from here) and Crypto- interaction under CO2-mediated low pH. Species monas pyrenoidifera Geitler (single flagellated differences in responses could lead to a shift in INTRODUCTION Elevated CO2 concentration will result in a cells – C. pyrenoidifera from here) were used as the community with a great ecological concern. decrease in pH which might affect intracellular testing organisms due to the fact they are easy to Besides conventional light-inverted microscopy, It is expected that atmospheric carbon dioxide pH, altering many physiological and structural handle and culture under experimental conditions flow cytometry is proposed as a potential tool for (CO2) will double over the next one hundred processes (Segovia et al., 2018). Moreover, it is and to the existence of previous information. Both phytoplankton optical properties analysis in this years (Solomon et al., 2007). Carbon capture and well documented that cells grown under high species are widely distributed around the world context of carbon capture and sequestration geological storage (CCS) technologies are among CO2 conditions have a less developed or an (Cepák et al., 2007; Guiry & Guiry, 2018) and environmental risk assessment, which allows rapid the carbon mitigation strategies being currently undetectable pyrenoid (Miyachi et al., 1986). sometimes in coexistence, e.g. in a reservoir in analyses of the growth rate and cell features and had discussed (IPCC, 2015; Szalaj et al., 2017). Many Besides, Morita et al. (1998) demonstrated Turkey (Sevindik, 2010) and in a subtropical lake previously been used in the monitoring of the physi- studies have been carried out regarding the risk of with non-pyrenoids algae that the presence of in China (Chen et al., 2008). ological state of microalgal cells (Cid et al., 1996). leakage from the delivery and geological storage pyrenoids or the accumulation of RuBisCO in of large volumes of CO2 (e.g. Basallote et al., chloroplasts is not always essential for the carbon 2012), where pH might be even lower than 4 pH concentration mechanism (CCM). Therefore, units (IPCC, 2005) and cause potential environ- physiological and fine structure properties of the mental consequences. Such acidification might cells could undergo changes in high CO2 concen- affect populations that inhabit the environment tration. These changes in the inner features of the around the pipe where the leakage happens. Envi- cell can be detectable by flow cytometry such as ronmental impacts on aquifers and in their micro- granularity (Shapiro, 1995). On the other hand, bial community have been described (Morozova the reduction of colony formation ability in S. et al., 2010), as well as hydro-chemical changes obliquus has been described under acidification associated with the upward migration of stored adjusted with HCl- (Yang et al., 2016) and CO2 through faults, fractures and poorly sealed or CO2-enhanced concentration (Huang et al., abandoned wells into shallow drinking water 2017). This was also reported in the species aquifers (Yang et al., 2014). The aquifer Botryococcus braunii when the size of the colony recharge-discharge is a relevant part of the suffers changes under high CO2 concentration hydrology of aquatic systems and CO2 leakages (Ge et al., 2011). This inducible defence plays an could have consequences on them. important role in preventing two important Microalgae are responsible for most primary ecological challenges faced by the microalgae, production in aquatic ecosystems and fuel energy sedimentation and grazing rates (Verschoor et al., transfer for the rest of the food web of these 2004; Stap et al., 2006). Thus, studies to reveal ecosystems, including the microbial loop (Cairns the effect of high CO2 concentration on the et al., 1992;). The pH of water affects algal colony formation of microalgae are necessary. growth and survival (e.g. Gensemer et al., 2018). The working hypothesis is that a low pH, The first studies carried out in the last third of the caused by a high concentration of CO2 in the twentieth century suggested that the high CO2 medium will affect microalgae at three levels: concentration in the medium would reduce the physiological, morphological and population efficiency in CO2 fixation by microalgae (Wer- levels (e.g. colony feature changes), that could dan et al., 1975; Coleman & Colman, 1981). lead to ecological consequences. In order to check Figure 1. Correlations between: (a) FSC-H Geo Mean values (cells size) and SSC-H Geo Mean values (granularity); (b) Red fluores- Primary production is expected to be stimulated this hypothesis, we developed laboratory-based cence (PerCP-Cy5-5-H Geo Mean) values and SSC-H Geo Mean values (granularity) of S. obliquus in monoculture. (c) FSC-H Geo in the short-term by the CO2 increase (Hein & manipulative experiments, which aimed to Mean values (cells size) and SSC-H Geo Mean values (granularity); (d) Red fluorescence (PerCP-Cy5-5-H Geo Mean) values and Sand-Jensen, 1997), so that one of the most disclose the effects of low pH caused by an injec- SSC-H Geo Mean values (granularity) of C. pyrenoidifera in monoculture. Correlaciones entre: (a) Valores de FSC-H Geo Mean (tamaños celulares) y SSC-H Geo Mean (granularidad); (b) Valores de fluorescencia roja (PerCP-Cy5-5-H Geo Mean) y SSC-H Geo prominent concerns about increased CO2 is that it tion of CO2 on the growth rates and cellular Mean values (granularidad) de S. obliquus en el monocultivo. (c) Valores de FSC-H Geo Mean (tamaños celulares) y valores de would induce uncontrollable primary production features in two microalgae used as experimental SSC-H Geo Mean (granularidad); (d) Valores de fluorescencia roja (PerCP-Cy5-5-H Geo Mean) y SSC-H Geo Mean (granularidad) (see the review by Riebesell & Tortell, 2011). models. The selected endpoints were Cell density, de C. pyrenoidifera en el monocultivo.

Limnetica, 37(2): 267-281 (2018)

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Carbon capture and well documented that cells grown under high species are widely distributed around the world context of carbon capture and sequestration geological storage (CCS) technologies are among CO2 conditions have a less developed or an (Cepák et al., 2007; Guiry & Guiry, 2018) and environmental risk assessment, which allows rapid the carbon mitigation strategies being currently undetectable pyrenoid (Miyachi et al., 1986). sometimes in coexistence, e.g. in a reservoir in analyses of the growth rate and cell features and had discussed (IPCC, 2015; Szalaj et al., 2017). Many Besides, Morita et al. (1998) demonstrated Turkey (Sevindik, 2010) and in a subtropical lake previously been used in the monitoring of the physi- studies have been carried out regarding the risk of with non-pyrenoids algae that the presence of in China (Chen et al., 2008). ological state of microalgal cells (Cid et al., 1996). leakage from the delivery and geological storage pyrenoids or the accumulation of RuBisCO in of large volumes of CO2 (e.g. Basallote et al., chloroplasts is not always essential for the carbon 2012), where pH might be even lower than 4 pH concentration mechanism (CCM). Therefore, units (IPCC, 2005) and cause potential environ- physiological and fine structure properties of the mental consequences. Such acidification might cells could undergo changes in high CO2 concen- affect populations that inhabit the environment tration. These changes in the inner features of the around the pipe where the leakage happens. Envi- cell can be detectable by flow cytometry such as ronmental impacts on aquifers and in their micro- granularity (Shapiro, 1995). On the other hand, bial community have been described (Morozova the reduction of colony formation ability in S. et al., 2010), as well as hydro-chemical changes obliquus has been described under acidification associated with the upward migration of stored adjusted with HCl- (Yang et al., 2016) and CO2 through faults, fractures and poorly sealed or CO2-enhanced concentration (Huang et al., abandoned wells into shallow drinking water 2017). This was also reported in the species aquifers (Yang et al., 2014). The aquifer Botryococcus braunii when the size of the colony recharge-discharge is a relevant part of the suffers changes under high CO2 concentration hydrology of aquatic systems and CO2 leakages (Ge et al., 2011). This inducible defence plays an could have consequences on them. important role in preventing two important Microalgae are responsible for most primary ecological challenges faced by the microalgae, production in aquatic ecosystems and fuel energy sedimentation and grazing rates (Verschoor et al., transfer for the rest of the food web of these 2004; Stap et al., 2006). Thus, studies to reveal ecosystems, including the microbial loop (Cairns the effect of high CO2 concentration on the et al., 1992;). The pH of water affects algal colony formation of microalgae are necessary. growth and survival (e.g. Gensemer et al., 2018). The working hypothesis is that a low pH, The first studies carried out in the last third of the caused by a high concentration of CO2 in the twentieth century suggested that the high CO2 medium will affect microalgae at three levels: concentration in the medium would reduce the physiological, morphological and population efficiency in CO2 fixation by microalgae (Wer- levels (e.g. colony feature changes), that could dan et al., 1975; Coleman & Colman, 1981). lead to ecological consequences. In order to check Primary production is expected to be stimulated this hypothesis, we developed laboratory-based in the short-term by the CO2 increase (Hein & manipulative experiments, which aimed to Sand-Jensen, 1997), so that one of the most disclose the effects of low pH caused by an injec- prominent concerns about increased CO2 is that it tion of CO2 on the growth rates and cellular would induce uncontrollable primary production features in two microalgae used as experimental (see the review by Riebesell & Tortell, 2011). models. The selected endpoints were Cell density,

270 Galotti et al.

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272 Galotti et al.

Table 1. Results of the repeated-measures ANOVA, using day in the experiment (time) as the within-subject factor and CO2 treatment S. obliquus growth rate (GR) in the monocul- (PerCP-Cy5-5-H geometric mean = red fluores- as the between-subject factor. Columns indicate the variables of interest, the degrees of freedom (d.f.1 and d.f.2), the value of the ture was significantly higher in the low-pH than in cence) and granularity (SSC-H geometric means) F-statistic (F) and the corresponding probability (p). Resultados de ANOVA de medidas repetidas utilizando los días en el experimento the control treatment (See Table 1). The biggest (Fig. 1b). The correlations between cell size and (tiempo) como factores intra-sujetos y el tratamiento con CO como factor entre-sujetos. Las columnas indican las variables de 2 difference between GR in low-pH and control granularity in S. obliquus monoculture were simi- interés, grados de libertad (d.f.1 y d.f.2), el valor del estadístico F (F) y la probabilidad correspondiente (p). treatments was seen on day 2 (See Table S1). lar for both the control and the low-pH treatments The biggest differences in cell size (FSC-H (F1,19= 166.368; p= 0.000; r2= 0.8986 for the 2 d.f.1 d.f.2 F p Geo Mean value) between treatment and control in control and F1,25= 247.314; p= 0.000; r = 0.8947

S. obliquus Cell Density within-subject 1 7 73524.252 0.000 monoculture were found on day 1 (See Table 1). for the low-pH treatment; Fig. 1a). There was a

monoculture between-subject 1 7 19.067 0.003 However, no significant differences were found correlation between chlorophyll and granularity

Growth Rate within-subject 1 7 132.898 0.000 after 7 days of the experiment (See Table 1). in the control treatment (F1,18= 61.575; p= 0.000; 2 between-subject 1 7 9.642 0.017 The oscillatory fashion of granularity was similar r = 0.7789), while there was not under low-pH 2 Cell Size within-subject 1 7 1729693.215 0.000 between the control and the low-pH treatments (p> 0.05; r = 0.0361) (Fig. 1b).

between-subject 1 7 72.979 0.000 during the experimental period. Values of granulari- The percentage of colonies in relation to ty were only higher in the control than in the low-pH single cells of S. obliquus decreased in the Granularity within-subject 1 7 965490.399 0.000 treatment on days 1 and 2 (See Table S1). low-pH treatments of monoculture (See Table between-subject 1 7 3.277 0.113 In order to assess that granularity is related to S1) and was significantly different from control Colony formation within-subject 1 14 17962.258 0.000 changes in inner features rather than to cell size (See Table 1), indicating lower colony formation between-subject 3 14 980.758 0.000 only, two correlations were performed. The first ability under 6.5 pH. C. pyrenoidifera Cell Density within-subject 1 3 154090.764 0.000 one between cell size (FSC-H geometric means) To confirm the low colony formation in monoculture between-subject 1 3 2.577 0.207 and granularity (SSC-H geometric means) (Fig. low-pH treatment, flow cytometry cytograms Growth Rate within-subject 1 2 1459.294 0.001 1a), and the second between chlorophyll were used. Figure 2 illustrates the response in between-subject 1 2 10.742 0.082

Cell Size within-subject 1 3 1328801.449 0.000

between-subject 1 3 41.449 0.008

Granularity within-subject 1 3 972096.446 0.000

between-subject 1 3 0.930 0.406

S. obliquus Cell Density within-subject 1 5 176689.919 0.000

competition between-subject 1 5 12.576 0.016

culture Growth Rate within-subject 1 5 625.275 0.000

between-subject 1 5 23.128 0.005

Cell Size within-subject 1 5 7353.439 0.000

between-subject 1 5 0.633 0.462

Granularity within-subject 1 5 536245.451 0.000

between-subject 1 5 1.729 0.246

Colony formation within-subject 1 4 12697.355 0.000

between-subject 3 4 1695.139 0.000

Total Biovolume within-subject 1 5 302929.250 0.000

between-subject 1 5 12.576 0.016

C. pyrenoidifera Cell Density within-subject 1 5 90878.145 0.000

competition between-subject 1 5 0.170 0.698

culture Growth Rate within-subject 1 5 104.789 0.000

between-subject 1 5 3.224 0.133

Cell Size within-subject 1 5 143214.974 0.000

between-subject 1 5 0.386 0.561

Granularity within-subject 1 5 555327.095 0.000

between-subject 1 5 26.163 0.004

Total Biovolume within-subject 1 5 232660.812 0.000

between-subject 1 5 0.221 0.658

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CO2-mediated low pH affects microalgae 273

Figure 2. The accumulated approximated biovolume (μm3· 106) by each FSC-H actual values (not channels from zero to 1024). Arrows highlight the inflection points on the curves. El biovolumen aproximado acumulado (μm3· 106) para cada valor real de FSC-H (no canales del cero al 1024). Las flechas apuntan los puntos de inflexión de las curvas.

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274 Galotti et al. accumulated approximated biovolume (µm3) in fashion in the control and low-pH treatments, each treatment, considering the number of cells in always in the contrary manner between treatments each FSC channel multiplied by the calibrated (See Table S2). Granularity was correlated with size of each channel. As in the accumulative cell size and chlorophyll content (Fig. 1c and d, probability curves, this representation allows the respectively). A significant negative correlation detection of deviation from normal distributions between granularity and red fluorescence (chloro- in dense continuous dot clusters. The control phyll content) was only observed in the low-pH curve showed two inflection points that corre- treatment (F1,16= 22.323; p= 0.000; r2= 0.6002). sponded to two subpopulations (singles and colony) within the cluster of this species. How- Competition experiment ever, in the low-pH treatment the accumulated curve showed a single inflection point, at the As expected, the cell density of S. obliquus and level of the smaller subpopulation. C. pyrenoidifera was significantly different between species throughout the experimental C. pyrenoidifera monoculture period, since the initial cell density was already different in order to accomplish similarities in C. pyrenoidifera had its growth stimulated in the total biovolume between species. However, only low-pH. Cell density was significantly lower in S. obliquus cell density was statistically different control (p> 0.05; See Table 1) on day 14. By the between treatments, low-pH and control (See end of the experiment, the cell density in the Table 1). S. obliquus cell density was lower low-pH treatment recovery was 28.5 % higher under the low-pH treatment of the competition than in the control on day 14 (Table S2). experiment than in monoculture. Meanwhile, C. The C. pyrenoidifera growth rate (d-1) was not pyrenoidifera cell density under low-pH was significantly different between treatments (See similar in both experiments. Table 1), and only slight differences were detected At the end of the competition experiment, S. during the initial days (day 1 and 2; See Table S2). obliquus growth rate was higher under low-pH Significant differences were seen on days 7, than control but lower in both cases than those in 10 and 14 when the cell size in the low-pH treat- the monoculture. Differences were significant ment was bigger than in the control (see Table S2; both between and within subjects (See Table 1). and Table 1 for significance levels). The C. pyrenoidifera growth rate was higher C. pyrenoidifera granularity (SSC-H geomet- under low-pH than the control at the end of the ric mean) responded in a somewhat oscillatory competition experiment, being even higher than the growth rates in both treatments in the mono- mentation, ending larger than on the initial day. In culture (See Table S2 and S4). Responses were addition, it was larger in the control than in the significantly different among species and treat- low-pH treatment (See Table S3 and Table 1). ments on day 2, day 10 and day 14 (p< 0.05). In the competition experiment, the response of In order to compare the growth rates between C. pyrenoidifera cell size observed between treat- monoculture and the competition experiment, ments was similar to monoculture, increasing the growth rate variation percentage was calcu- both in the low-pH and control but higher in the lated (see Fig. 3). The highest percentage of low-pH treatment as well as in the monoculture variation was observed on the first day of exper- (See Table S4). imentation for both species, S. obliquus (Fig. 3a) S. obliquus granularity (SSC-H values) has and C. pyrenoidifera (Fig. 3b). From the second shown the same pattern during the experiment under day, the variation in S. obliquus remained nega- the low-pH and control treatments (See Table S3), tive in the control and the low-pH treatments. while C. pyrenoidifera granularity was higher in the The variation percentage in C. pyrenoidifera low-pH treatment than in the control treatment, Figure 3. Growth rate variation (%) in the competition experiment in relation to the monoculture under low-pH and control treatments remained positive from day 7. especially on days 7 and 10 (See Table S4). The for S. obliquus (a) and C. pyrenoidifera (b). Porcentaje de variación de la tasa de crecimiento en el experimento de competición en The cell size of S. obliquus responded in an difference throughout the period of the experiment relación al monocultivo en el tratamiento con bajo pH y control para S. obliquus (a) y C. pyrenoidifera (b). oscillatory fashion during the period of experi- was significant for both species (See Table 1).

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CO2-mediated low pH affects microalgae 275 accumulated approximated biovolume (µm3) in fashion in the control and low-pH treatments, each treatment, considering the number of cells in always in the contrary manner between treatments each FSC channel multiplied by the calibrated (See Table S2). Granularity was correlated with size of each channel. As in the accumulative cell size and chlorophyll content (Fig. 1c and d, probability curves, this representation allows the respectively). A significant negative correlation detection of deviation from normal distributions between granularity and red fluorescence (chloro- in dense continuous dot clusters. The control phyll content) was only observed in the low-pH curve showed two inflection points that corre- treatment (F1,16= 22.323; p= 0.000; r2= 0.6002). sponded to two subpopulations (singles and colony) within the cluster of this species. How- Competition experiment ever, in the low-pH treatment the accumulated curve showed a single inflection point, at the As expected, the cell density of S. obliquus and level of the smaller subpopulation. C. pyrenoidifera was significantly different between species throughout the experimental C. pyrenoidifera monoculture period, since the initial cell density was already different in order to accomplish similarities in C. pyrenoidifera had its growth stimulated in the total biovolume between species. However, only low-pH. Cell density was significantly lower in S. obliquus cell density was statistically different control (p> 0.05; See Table 1) on day 14. By the between treatments, low-pH and control (See end of the experiment, the cell density in the Table 1). S. obliquus cell density was lower low-pH treatment recovery was 28.5 % higher under the low-pH treatment of the competition than in the control on day 14 (Table S2). experiment than in monoculture. Meanwhile, C. The C. pyrenoidifera growth rate (d-1) was not pyrenoidifera cell density under low-pH was significantly different between treatments (See similar in both experiments. Figure 4. Correlations between: (a) FSC-H Geo Mean values (cells size) and SSC-H Geo Mean values (granularity); (b) Red fluores- Table 1), and only slight differences were detected At the end of the competition experiment, S. cence (PerCP-Cy5-5-H Geo Mean) values and SSC-H Geo Mean values (granularity) of S. obliquus in the competition experiment. (c) during the initial days (day 1 and 2; See Table S2). obliquus growth rate was higher under low-pH FSC-H Geo Mean values (cells size) and SSC-H Geo Mean values (granularity); (d) Red fluorescence (PerCP-Cy5-5-H Geo Mean) Significant differences were seen on days 7, than control but lower in both cases than those in values and SSC-H Geo Mean values (granularity) of C. pyrenoidifera in the competition experiment. Correlaciones entre: (a) Valores de FSC-H Geo Mean (tamaños celulares) y SSC-H Geo Mean (granularidad); (b) Valores de fluorescencia roja (PerCP-Cy5-5-H Geo 10 and 14 when the cell size in the low-pH treat- the monoculture. Differences were significant Mean) y SSC-H Geo Mean values (granularidad) de S. obliquus en el experimento de competición. (c) Valores de FSC-H Geo Mean ment was bigger than in the control (see Table S2; both between and within subjects (See Table 1). (tamaños celulares) y valores de SSC-H Geo Mean (granularidad); (d) Valores de fluorescencia roja (PerCP-Cy5-5-H Geo Mean) y and Table 1 for significance levels). The C. pyrenoidifera growth rate was higher SSC- H Geo Mean (granularidad) de C. pyrenoidifera en el experimento de competición. C. pyrenoidifera granularity (SSC-H geomet- under low-pH than the control at the end of the ric mean) responded in a somewhat oscillatory competition experiment, being even higher than the growth rates in both treatments in the mono- mentation, ending larger than on the initial day. In culture (See Table S2 and S4). Responses were addition, it was larger in the control than in the significantly different among species and treat- low-pH treatment (See Table S3 and Table 1). ments on day 2, day 10 and day 14 (p< 0.05). In the competition experiment, the response of In order to compare the growth rates between C. pyrenoidifera cell size observed between treat- monoculture and the competition experiment, ments was similar to monoculture, increasing the growth rate variation percentage was calcu- both in the low-pH and control but higher in the lated (see Fig. 3). The highest percentage of low-pH treatment as well as in the monoculture variation was observed on the first day of exper- (See Table S4). imentation for both species, S. obliquus (Fig. 3a) S. obliquus granularity (SSC-H values) has and C. pyrenoidifera (Fig. 3b). From the second shown the same pattern during the experiment under day, the variation in S. obliquus remained nega- the low-pH and control treatments (See Table S3), tive in the control and the low-pH treatments. while C. pyrenoidifera granularity was higher in the The variation percentage in C. pyrenoidifera low-pH treatment than in the control treatment, remained positive from day 7. especially on days 7 and 10 (See Table S4). The The cell size of S. obliquus responded in an difference throughout the period of the experiment oscillatory fashion during the period of experi- was significant for both species (See Table 1).

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278 Galotti et al. increased but not in response to an increment of production of biomass and biocompounds by of CO2. Bioresource Technology, 136: acuáticos salinos del alto Guadalquivir. chlorophyll content, contrary to what happens in three different microalgae. Engineering in 496–501. DOI: 10.1016/j.biortech.2013.03.072 Limnetica, 25: 763-770. C. pyrenoidifera under low-pH. It confirms the Life Sciences, 17: 1126–1135. DOI: CHOIX, F. J., C. G. LÓPEZ-CISNEROS & H. O. GE, Y., J. LIU & G. TIAN. 2011. Growth charac- results taken by Yang & Gao (2003) when they 10.1002/elsc.201700075 MÉNDEZ-ACOSTA. 2018. Azospirillum teristics of Botryococcus braunii 765 under found that the photosynthetic physiology of S. BASALLOTE, M.D., A. RODRÍGUEZ-ROME- brasilense increases CO2 fixation on microal- high CO2 concentration in photobioreactor. obliquus was affected by high CO2 concentra- RO, J. BLASCO, A. DELVALLS & I. RIBA. gae Scenedesmus obliquus, Chlorella vulgaris, Bioresource Technology, 102: 130-134. DOI: tions. S. obliquus physiology was not only affect- 2012. Lethal effects on different marine and Chlamydomonas reinhardtii cultured on 10.1016/j.biortech.2010.06.051 ed in this way, because the CO2-mediated low pH organisms, associated with sediment-seawater high CO2 concentrations. Microbial Ecology, GENSEMER, R. W., J. C. GONDEK, P. H. also minimizes its ability to form colonies. acidification deriving from CO2 leakage. Envi- 1-13. DOI: 10.1007/s00248-017-1139-z RODRIQUEZ, J. J. ARBILDUA, W. A. Regarding flow cytometry, it responded as a ronmental Science and Pollution Research, 19: CID, A., P. FIDALGO, C. HERRERO & J. STUBBLEFIELD, A. S. CARDWELL, R. C. potent tool capable of analysing thousands of cells 2550-2560. DOI: 10.1007/s11356-012-0899-8 ABALDE. 1996. Toxic action of copper on SANTORE, A. C. RYAN, W.J. ADAMS, E. in seconds. It was disclosed as a sensitive and BAUTISTA-CHAMIZO, E., M. R. DE ORTE, T. the membrane system of a marine diatom mea- NORDHEIM & NORDHEIM, E. 2018. Eval- rapid technique in the present study being deter- Á. DELVALLS & I. RIBA. 2016. Simulating sured by flow cytometry. Cytometry, 25: 32-36. uating the effects of pH, hardness, and minant in providing quick and reliable results. CO2 leakages from CCS to determine Zn toxic- DOI: 10.1002/(SICI)1097-0320(19960901) dissolved organic carbon on the toxicity of In conclusion, the elevated CO2 concentration ity using the marine microalgae Pleurochrysis 25:1<32::AID-CYTO4>3.0.CO;2-G aluminum to freshwater aquatic organisms modified the growth dynamics and specific cell roscoffensis. Chemosphere, 144: 955-965. COLEMAN, J. R. & B. COLMAN. 1981. under circumneutral conditions. 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CO2-mediated low pH affects microalgae 279 increased but not in response to an increment of production of biomass and biocompounds by of CO2. Bioresource Technology, 136: acuáticos salinos del alto Guadalquivir. chlorophyll content, contrary to what happens in three different microalgae. Engineering in 496–501. DOI: 10.1016/j.biortech.2013.03.072 Limnetica, 25: 763-770. C. pyrenoidifera under low-pH. It confirms the Life Sciences, 17: 1126–1135. DOI: CHOIX, F. J., C. G. LÓPEZ-CISNEROS & H. O. GE, Y., J. LIU & G. TIAN. 2011. Growth charac- results taken by Yang & Gao (2003) when they 10.1002/elsc.201700075 MÉNDEZ-ACOSTA. 2018. Azospirillum teristics of Botryococcus braunii 765 under found that the photosynthetic physiology of S. BASALLOTE, M.D., A. RODRÍGUEZ-ROME- brasilense increases CO2 fixation on microal- high CO2 concentration in photobioreactor. obliquus was affected by high CO2 concentra- RO, J. BLASCO, A. DELVALLS & I. 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It was disclosed as a sensitive and BAUTISTA-CHAMIZO, E., M. R. DE ORTE, T. the membrane system of a marine diatom mea- NORDHEIM & NORDHEIM, E. 2018. Eval- rapid technique in the present study being deter- Á. DELVALLS & I. RIBA. 2016. Simulating sured by flow cytometry. Cytometry, 25: 32-36. uating the effects of pH, hardness, and minant in providing quick and reliable results. CO2 leakages from CCS to determine Zn toxic- DOI: 10.1002/(SICI)1097-0320(19960901) dissolved organic carbon on the toxicity of In conclusion, the elevated CO2 concentration ity using the marine microalgae Pleurochrysis 25:1<32::AID-CYTO4>3.0.CO;2-G aluminum to freshwater aquatic organisms modified the growth dynamics and specific cell roscoffensis. Chemosphere, 144: 955-965. COLEMAN, J. R. & B. COLMAN. 1981. under circumneutral conditions. 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Aquatic Sciences: 68: light–dark cycles on growth, productivity, and (CTM2012-36476-C02-02; co-funded by the Pérez-Ramírez (eds.): 63–89. John Wiley & 425-433. DOI: 10.1007/s00027-009-9143-0 biochemical composition. Applied biochemis- European Regional Development Fund). Our Sons Ltd, West Sussex. UK. DOI: FRANKLIN, N. M., M. S. ADAMS, J. L. STAU- try and biotechnology, 172: 2377-2389. DOI: thanks go to the laboratories that provided us with 10.1002/9781119154051.ch4 BER & R. P. LIM. 2001. Development of an 10.1007/s12010-013-0679-z the algal cultures, the Chemical Engineering CAIRNS, JR. J., P. MCCORMICK & S. improved rapid enzyme inhibition bioassay GUIRY, M.D. & G.M. GUIRY. 2018. Algae- Laboratory (University of Jaén, Spain) and the BELANGER. 1992. Ecotoxicological testing: with marine and freshwater microalgae using Base. World-wide electronic publication, Water Institute (University of Granada, Spain). small is reliable. Journal of Enviromental flow cytometry. Archives of Environmetal National University of Ireland, Galway. Technical and human support provided by CICT Pathology and Toxicology, 11: 247-263. Contamination and Toxicology, 40: 469-480. http://www.algaebase.org; searched on 05 of Universidad de Jaén (UJA, Ministerio de CAMPBELL, J. W. 1995. The lognormal distribu- DOI: 10.1007/s002440010199 February 2018. Economía y Competividad, Junta de Andalucía, tion as a model for bio‐optical variability in the FRANQUEIRA, D., A. CID, E. TORRES, M. HEIN, M. & K. SAND-JENSEN. 1997. CO2 European Regional Development Fund) is grate- sea. Journal of Geophysical Research-Oceans, OROSA & C. HERRERO. 1999. A compari- increases oceanic primary production. Nature, fully acknowledged. Undoubtedly, our thanks are 100: 13237-13254. DOI: 10.1029/95JC00458 son of the relative sensitivity of structural 388:526-527. DOI: 10.1038/41457 also due to the editor of this Journal and to the two CEPÁK, V., P. PRIBYL, M. 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Limnetica, 37(2): 267-281 (2018) Growth rate, Cell size, Granularity, Single/colony For this purpose, monospecific culture experi- cell percentage and Biovolume. The cosmopolitan ments and a competition experiment were carried freshwater microalga, Scenedesmus (Acutodesmus) out with both species for 14 days. The competi- obliquus (Turpin) Kützing (single cells and tion experiment tries to clarify the interspecific coenobia – S. obliquus from here) and Crypto- interaction under CO2-mediated low pH. Species monas pyrenoidifera Geitler (single flagellated differences in responses could lead to a shift in INTRODUCTION Elevated CO2 concentration will result in a cells – C. pyrenoidifera from here) were used as the community with a great ecological concern. decrease in pH which might affect intracellular testing organisms due to the fact they are easy to Besides conventional light-inverted microscopy, It is expected that atmospheric carbon dioxide pH, altering many physiological and structural handle and culture under experimental conditions flow cytometry is proposed as a potential tool for (CO2) will double over the next one hundred processes (Segovia et al., 2018). Moreover, it is and to the existence of previous information. Both phytoplankton optical properties analysis in this years (Solomon et al., 2007). Carbon capture and well documented that cells grown under high species are widely distributed around the world context of carbon capture and sequestration geological storage (CCS) technologies are among CO2 conditions have a less developed or an (Cepák et al., 2007; Guiry & Guiry, 2018) and environmental risk assessment, which allows rapid the carbon mitigation strategies being currently undetectable pyrenoid (Miyachi et al., 1986). sometimes in coexistence, e.g. in a reservoir in analyses of the growth rate and cell features and had discussed (IPCC, 2015; Szalaj et al., 2017). Many Besides, Morita et al. (1998) demonstrated Turkey (Sevindik, 2010) and in a subtropical lake previously been used in the monitoring of the physi- studies have been carried out regarding the risk of with non-pyrenoids algae that the presence of in China (Chen et al., 2008). ological state of microalgal cells (Cid et al., 1996). leakage from the delivery and geological storage pyrenoids or the accumulation of RuBisCO in of large volumes of CO2 (e.g. Basallote et al., chloroplasts is not always essential for the carbon 2012), where pH might be even lower than 4 pH concentration mechanism (CCM). Therefore, units (IPCC, 2005) and cause potential environ- physiological and fine structure properties of the mental consequences. Such acidification might cells could undergo changes in high CO2 concen- affect populations that inhabit the environment tration. These changes in the inner features of the around the pipe where the leakage happens. Envi- cell can be detectable by flow cytometry such as ronmental impacts on aquifers and in their micro- granularity (Shapiro, 1995). On the other hand, bial community have been described (Morozova the reduction of colony formation ability in S. et al., 2010), as well as hydro-chemical changes obliquus has been described under acidification associated with the upward migration of stored adjusted with HCl- (Yang et al., 2016) and CO2 through faults, fractures and poorly sealed or CO2-enhanced concentration (Huang et al., abandoned wells into shallow drinking water 2017). This was also reported in the species aquifers (Yang et al., 2014). The aquifer Botryococcus braunii when the size of the colony recharge-discharge is a relevant part of the suffers changes under high CO2 concentration hydrology of aquatic systems and CO2 leakages (Ge et al., 2011). This inducible defence plays an could have consequences on them. important role in preventing two important Microalgae are responsible for most primary ecological challenges faced by the microalgae, production in aquatic ecosystems and fuel energy sedimentation and grazing rates (Verschoor et al., transfer for the rest of the food web of these 2004; Stap et al., 2006). Thus, studies to reveal ecosystems, including the microbial loop (Cairns the effect of high CO2 concentration on the et al., 1992;). The pH of water affects algal colony formation of microalgae are necessary. growth and survival (e.g. Gensemer et al., 2018). The working hypothesis is that a low pH, The first studies carried out in the last third of the caused by a high concentration of CO2 in the twentieth century suggested that the high CO2 medium will affect microalgae at three levels: concentration in the medium would reduce the physiological, morphological and population efficiency in CO2 fixation by microalgae (Wer- levels (e.g. colony feature changes), that could dan et al., 1975; Coleman & Colman, 1981). lead to ecological consequences. In order to check Primary production is expected to be stimulated this hypothesis, we developed laboratory-based in the short-term by the CO2 increase (Hein & manipulative experiments, which aimed to Sand-Jensen, 1997), so that one of the most disclose the effects of low pH caused by an injec- prominent concerns about increased CO2 is that it tion of CO2 on the growth rates and cellular would induce uncontrollable primary production features in two microalgae used as experimental (see the review by Riebesell & Tortell, 2011). models. The selected endpoints were Cell density,

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