The relationship between calcification and photosynthesis in the coccolithophorid Pleurochrysis carterae

Acta Ecologica Sinica 32 (2012) 38–43

Contents lists available at SciVerse ScienceDirect

Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes

The relationship between calcification and photosynthesis in the coccolithophorid Pleurochrysis carterae Zhou Chengxu ⇑, Jiang Ying, Liu Baoning, Yan Xiaojun, Zhang Wendong Marine Biotechnology Key Laboratory, Ningbo University, Ningbo 315211, Zhejiang, China

a r t i c l e

i n f o

Keywords: Pleurochrysis carterae Calcification Photosynthesis Coccolithophorids

a b s t r a c t Coccolithophorids are one of the dominant groups of marine phytoplankton. They are found in large numbers throughout the surface euphotic zone of the ocean, and are able to form large-scale blooms that persist for long periods of time. Coccolithophorid cells are covered by species-specific calcium carbonate crystals of various structures. In the process of calcification in coccolithophorids, Ca2+ is absorbed into cells from the culture medium, and a coccolith unit is formed inside the cell. Then, the coccolith unit extrudes to the cell surface where it is constructed into crystal layers. The formation of these crystals is regulated by cellular metabolism under different environmental conditions. The carbon biogeochemical cycle in the coccolithophorids involves both photosynthetic and calcification processes, which not only play an important role in population dynamics, but also in the global carbon cycle and climate change. However, one important question remains, namely, whether the relationship between photosynthesis and calcification is species-dependent. Previous studies have yielded controversial results, even in the same species. In this paper, we selected Pleurochrysis carterae, a coccolithophore species that frequently blooms in coastal areas, to study the relationship between calcification and photosynthesis. First, we studied population growth in a batch culture over several days. For batch cultures, P. carterae was inoculated into a 10 L bioreactor at an initial cell density of approximately 5  104 cells mL1. The culture conditions were optimal for cell growth. Dissolved oxygen (DO) was detected during all the culture period, and the rate of photosynthetic oxygen evolution was calculated according the DO changes during the 12-h illumination period. Algal samples (10 mL) were collected during the population growth phases. The calcium carbonate content on the cell surface was determined each day by chemical titration. Next, we studied the relationship between photosynthesis and calcification at the cellular level by observing patterns of recalcification during a 12-h period. In this study, non-calcified cells were obtained by decalcifying calcified cells collected during the exponential growth period in MES-NaOH buffer solution (pH 5.5). The non-calcified cells were inoculated into culture media containing different concentrations of Ca2+ (0, 5, 20, 40, 50, or 100 mg L1). The rate of recalcification was determined by microscopic analyses in which the number of recalcified cells per 100 cells was counted at 0, 3, 6, 9, and 12 h of culture. Ca2+ absorbed into the cell was detected by measuring the fluorescence intensity of Fluo-3/AM labeled Ca2+. The rate of photosynthetic oxygen evolution in the non-calcified cell cultures was detected by measuring the changes in dissolved oxygen during the 12-h illumination period. The results showed that during the population growth period, the rate of photosynthetic oxygen evolution was inversely related to the calcium carbonate content per cell. When the amount of calcium carbonate on the cell surface increased, the relative photosynthetic ability (the rate of photosynthetic oxygen evolution) decreased, and vice versa. Both recalcification rates and photosynthetic oxygen evolution were affected by the extracellular calcium concentration. Non-calcified cells showed different recalcification abilities at different extracellular Ca2+ concentrations. The recalcification rate of non-calcified cells was positively correlated with the extracellular calcium concentration when [Ca2+] in the medium ranged from 0 to 100 mg L1. However, photosynthetic oxygen evolution was suppressed at higher cell calcification rates, especially when extracellular [Ca2+] was 50–100 mg L1. Our analyses of the population growth process and the cell recalcification process confirmed that photosynthesis is inversely related to calcification in P. carterae. Ó 2011 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

⇑ Corresponding author. E-mail address: [email protected] (C. Zhou). 1872-2032/$ - see front matter Ó 2011 Ecological Society of China. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chnaes.2011.12.003

C. Zhou et al. / Acta Ecologica Sinica 32 (2012) 38–43

1. Introduction Coccolithophorids are one of the dominant groups of marine phytoplankton. They are found in large numbers throughout the euphotic zone of the ocean, and are able to form large-scale blooms that persist for long periods of time. The carbon biogeochemical cycle in the coccolithophorids involves both photosynthetic and biomineralization (calcification) processes, which can form species-specific calcium carbonate crystals of various structures covered on the cell surface, called coccoliths [1]. In the process of photosynthesis, coccolithophorids assimilate CO2 into organic compounds (CO2 + H2O ? CH2O + O2), whereas in the process of calcification, Ca2+ is absorbed into cells from the culture medium, and a coccolith unit is formed inside the cell, along with release of CO2 ðCa2þ þ 2HCO 3 ! CaCO3 þ H2 O þ CO2 Þ. Therefore, the relationship between calcification and photosynthesis in the coccolithophorid play an important role in environmental carbon source, carbon sink, global carbon cycle, and related climate changes [2]. Although no systematic analysis has been carried out, there are distinctive differences on the ratio of calcification/photosynthesis (C/P) depending on species, about 1 for Emiliania huxleyi and Cricosphaera carterae [3–5], 0.6–0.5 for Umbilicosphaera sibo and highly calcified Pleurochrysis sp. [6,7]. One important question remains, whether the relationship between photosynthesis and calcification is species-specific, and the effects on atmospheric carbon dioxide will have by the calcification/photosynthesis changes along with the coccolithophorid population dynamics. Previous studies have yielded controversial results on calcification capacity, environmental responses, the relationship between calcification and photosynthesis, even in the same species [8–11]. The focus point is that formation of coccolith by utilizing extra-cellular calcium ion and the extrusion of coccolith to the cell surface is not just a simple physico-chemical process, but a physiological process, the formation of these coccolith crystals is regulated by cellular metabolism under different environmental conditions [12]. For example, the calcification is gradually intensified with the growth period in Pleurochrysis carterae, the calcium level inside the cell or in the culture medium will affect the calcification capacity. However, no simultaneous analysis of photosynthesis was carried out [13]. In this paper, we selected P. carterae, a coccolithophore species that frequently blooms in coastal areas, to study the relationship between calcification and photosynthesis of this species. The cellular calcification efficiency, photosynethsis capacity are measured during the re-calcification in the function of different calcium levels, as well as during a whole growth period for P. carterae cultured in photobioreactor. 2. Materials and methods 2.1. Microalgal culture Pure strain of P. carterae was obtained from the Marine Biotechnology Laboratory of Ningbo University (strain number NMBjih026). Cultures were carried out in illumination incubator at 20 °C at an irradiance of 40 lmol photons m2 s1 in light dark cycle L:D of 12:12. The culture medium is NMB3 [14], and described briefly as follows: the seawater for culture was sterilized (pH 8.30, salinity 27‰) and enriched with nutrients: KNO3 100 mg L1, KH2PO4 10 mg L1, MnSO4H2O 0.25 mg L1, FeSO47H2O 2.50 mg L1, EDTA-Na2 10 mg L1, Vitamin B1 6 lg L1, Vitamin B12 0.05 lg L1. 2.2. Experimental methods 2.2.1. Cellular assay of photosynthesis and calcification 2.2.1.1. Preparation of culture medium with different calcium gradient. The calcium free artificial seawater was prepared according to

39

Harrison et al. [15]. The artificial seawater was enriched with the stock solution of NMB3 medium, and calcium chloride was added respectively. The calcium gradient was 0, 5, 20, 40, 50, 100 mg/L. 2.2.1.2. The collection of microalgal cells with different calcification degree. The calcified cells (C-cell) was collected at exponential phase, and the naked cells (N-cell) was obtained by treating those C-cells with MES-NaOH buffer (0.5 mol/L, pH 5.5) for 2 h to remove coccoliths. Then, the N-cells was collected by centrifugation (4000 r/min, 1 min), and washed with artificial seawater thrice. The N-cells were suspended in 96-well microplate with 200 lL culture medium with different calcium gradient, the cells with inoculation density of 106 cell/mL was cultured in illumination incubator at 20 °C at an irradiance of 40 lmol photons m2 s1 in light dark cycle L:D of 24:0. 15 replicates were prepared for each calcium gradient in the 96-well plate. The cells per 3 wells were observed under inverted microscope at time of 0, 3, 6, 9, 12 h respectively after fixation with formaldehyde. The ratio of re-calcified cells was recorded by counting 100 cells from each microplate well. 2.2.1.3. Free intracellular calcium measurement by Fluo-3/AM. Free calcium can bind Fluo-3 to form fluorescent compound quantitatively. Fluo-3/AM was used to detect the calcification process for P. carterae cells at different calcium gradient [16,17]. The N-cells (500 lL) was cultured in 2 mL Eppendorff tubes at 2  106 cells mL1, using 12 EP tubes for each gradient. The culture conditions were the same as Section 2.2.1.2. The dye of Fluo-3/AM was applied at 0 h, and 12 h, respectively, with six tubes for each time point. The fluorescence dyeing protocol was described briefly as follows: firstly, fixation was carried out using 1 drop of 5% formaldehyde solution for 30 min in each EP tube, after washing with the calcium free artificial seawater thrice, the cells was suspended in 0.01% Pluronic F-127 up to 500 lL, 10 lL 0.2 mM Fluo-3/AM was added with final concentration of 4 lM, the cells were incubated in darkness at 25 °C for 30 min, and then washed with calcium free artificial seawater thrice again, re-suspended again and incubated in darkness at 25 °C for another 24 h. 200 lL sample was sipped, and recorded in 96-well fluorescence microplate reader (Varioskan Flash, Thermo Scientific) with excitation wavelength of 488 nm, and emission wavelength of 526 nm. The fluorescence intensity was calculated as DF = F  F0 to reflect the intracellular calcium concentration, where F was read from the experimental group with addition of calcium solution, F0 was from the control group with calcium free artificial seawater. Each group was carried out in triplicate. 2.2.1.4. Net photosynthesis at different calcium gradient. Dissolved oxygen (DO) level was used to indicate the net photosynthesis. The N-cells were cultured in calcium free artificial seawater adding different calcium gradients. 10 mL N-cells in 25 mL flask, with inoculation density of 106 cell/mL, was cultured in illumination incubator at 20 °C at an irradiance of 40 lmol photons m2 s1 in light dark cycle L:D of 24:0 for 12 h. Chlorophyll a and dissolved oxygen were determined at 0 h, and 12 h, respectively [18]. Each group was carried out in triplicate. The net oxygen release rate was calculated as: DO1000 Net photosynthetic oxygen (lmol mg1 h1) ¼ D32Chlat

where Chl a (mg/L) averaged from the time point of 0 h and 12 h t = 12. 2.2.2. Monitoring of photosynthesis and calcification process during the population changes 2.2.2.1. Culture of P. carterae in photobioreactor. Cultures were inoculated into the 10-L air-lift photobioreactor with 6000 mL microalgal solution pre-cultured to exponential phase in flasks, 4000 mL

C. Zhou et al. / Acta Ecologica Sinica 32 (2012) 38–43

2.2.2.2. Measurement of cell surface calcification degree by EDTA complexometric titration method [19]. Calibration curve for calcium quantitation: Calibration was carried out in triplicate by using EDTA complexometric titration method for the authentic CaCl2 solution (100 mL) with concentration of 4, 6, 8, 10 lM. 1 mL NaOH (1.5 mM) and 0.002–0.02 Calconcarboxylic acid (Calcon indicator) was added respectively, the concentration of EDTA-Na2 was 0.5 mM. The equilibrium end point was recorded by observing until the color of Calcon indicator was changed from red to blue, and not reversed for 0.5 min. The regressive equation of calibration curve was: y = 1.646x, R2 = 0.999. Calcium content measurement in microalgal sample: The cells were collected by centrifugation (3000 r/min, 10 min) from 10 mL of microalgal culture, followed by decanting the supernatant. six replicates were prepared, three samples were adding 10 mL deionized water, and the other ones were adding 10 mL, 0.1 mol/L HCl, each measurement was carried out by EDTA complexometric titration. Calcium content was obtained from regressive calibration curve. Calculation of cell surface calcification degree: The calcium content in the above microalgal sample with addition of deionized water was accounted as the soluble calcium ion, the calcium content in the sample with addition of HCl was accounted as total calcium. The calcium content of cell surface carbonate crystal was obtained by substracting the soluble calcium ion from total calcium, which was the indication of cell surface calcification degree. 2.2.2.3. Net photosynthetic oxygen release during population dynamic period. The dissolved oxygen concentration in the photobioreactor was directly measured by DO meter (RSS-5100), recorded at 9:00 and 21:00, daily. The content of Chl a was measured daily. Net oxygen release in photobioreator based on Chl a was determined, using the same equation in Section 2.2.1.4. 2.3. Statistics Data were analyzed by single factor ANOVA (a = 0.05) using SPSS (11.5) [20]. 3. Results 3.1. Cellular assay of photosynthesis and calcification 3.1.1. Effects on re-calcification at different Ca2+ gradients Re-calcification of the N-cell of P. carterae at different Ca2+ gradients was shown in Fig. 1. It has been shown that the calcification rate was increased with the calcium gradient. There was no calcified cell (C-cell) in the calcium-free seawater medium. The re-calcification was initiated in the calcium-enriched medium, and can reach about 20% at 3 h. Then, the calcification process began to differentiate: for low calcium level (5 mg/mL), the cells maintained its calcification state only for 3 h, and de-calcify to the N-cells at 9 h; for moderate calcium levels (20, 40 mg/mL), the cells maintained its calcification state for 3 h, and increase its calcification rate to be 40%, and 65% at 12 h, respectively; for high calcium level (50, 100 mg/mL), the cells increased their re-calcification process continuously, and most of the cells changed to be C-cells with the rate higher than 80% in 12 h.

0mg/L

100

Calcification rate (%)

new culture medium enriched with NMB3 stock solution was added. This culture was grown at 20–25 °C at an irradiance of 90–100 lmol photons m2 s1 in light dark cycle L:D of 12:12, with aeration of 0.5 L min1. The cell density was monitored by haemocytometer daily.

5mg/L

80

20mg/L

60

40mg/L 50mg/L

40

100mg/L

20 0 0

3

6

9

12

Time (hours) Fig. 1. Rate of calcification at different Ca2+ concentrations for P. carterae. Error bars = +/ 1 SD and n = 3.

3.1.2. Changes of calcium fluorescent Fluo-3/AM assay at different Ca2+ gradients The intracellular calcium concentration was determined by calcium fluorescent Fluo-3/AM assay, and the results were given in Fig. 2. It was shown that the calcium level for all calcium groups was kept the same as ca. 0.5 AU at the beginning of the experiment (0 h), whereas the fluorescent intensity after 12 h was increased along with the calcium enrichment levels. However, the most significant value of DF was appeared at the highest calcium level of 100 mg/mL (P < 0.01), while only slight changes were appeared at low calcium levels such as 5 and 20 mg/mL. Combined the results of Figs. 1 and 2, it was indicated that the extracellular calcium level determined the intracellular calcium level, as well as the extrusion of coccoliths from cell surface. The higher the calcium concentration, the more the calcification rate. Therefore, it should be very feasible to fulfill the calcification control by providing different levels of extracellular calcium. 3.1.3. The net photosynthetic oxygen release at different Ca2+ gradients Oxygen release rate during the re-calcification process indicated that oxygen concentration maintained at the same level for the calcium levels from 0 to 40 mg/mL without significant differences (P > 0.05), and decreased 25% for the calcium levels from 50 to 100 mg/mL, significantly lower than the low calcium level groups (P < 0.01) (Fig. 3). It was indicated that the photosynthesis was largely inhibited in the high calcium medium. 3.1.4. Correlation among intracellular calcium level, calcification rate and photosynthetic oxygen release The correlation among photosynthetic oxygen release rate, intracellular calcium fluorescence intensity, and re-calcification rate of P. carterae cell was shown in Figs. 4 and 5, respectively. It was observed that photosynthesis and calcification was inversely correlated at the whole range of extracellular calcium concentration. The intracellu-

ΔF (A.U.)

40

4 3.5 3 2.5 2 1.5 1 0.5 0

0h 12 h

0

5

20 2+

40

50

100

-1

Ca concentration (mg.L ) Fig. 2. Fluorescence intensity for different degrees of calcification that were marked by Fluo-3/AM. Error bars = +/ 1 SD and n = 3.

Crystal calcium carbonate

3.0

Photosynthetic oxygen evolution

0 -0.5 -1 -1.5 -2

0

5

Ca

20 2+

40

50

100

-1

Fig. 3. Photosynthetic rate of P. carterae at different Ca concentrations. Error bars = +/ 1 SD and n = 3.

Photosynthetic O2 evolution (µmol O2/(mg Chla•h))

3.5

0

2.5 20

40

50

100

2

-0.3

1.5 1

-0.6

ΔF (A.U.)

3 5

0.5 -0.9

0

Ca2+ concentration (mg.L-1)

0-12 h Photosynthetic oxygen evolution 12 h Calcification rate

0.4

100

0.2

80

0 0

5

20

40

50

100

-0.4

60 40

-0.6 20

-0.8 -1

0 2+

Calcification rate (%)

Photosynthetic O2 evolution (µmol O 2/(mg Chla•h))

Fig. 4. Fluorescence intensity and photosynthetic rate of P. carterae cell for different degrees of calcification. Error bars = +/ 1 SD and n = 3.

-0.2

-1

Fig. 5. Calcification and photosynthetic rates at different Ca concentrations for P. carterae. Error bars = +/ 1 SD and n = 3.

4

-1

(×10 cells.mL )

Cell number

35 30 25 20 15 10 5 1

2

3

4

2.0

20

1.5 1.0

0

0.5 1

2

3

4

5

6

-20

Time (days) Fig. 7. Calcification and photosynthetic rates of P. carterae in batch culture. Error bars = +/ 1 SD and n = 3.

oxygen release rate was much more complex for different degrees of calcification. The net photosynthetic oxygen release was positive for the calcium level of 0–40 mg/mL, without significant differences (P > 0.05), but decreased to be negative when the calcium level higher than 50 mg/mL, an obvious indication of significant inhibition of photosynthesis. 3.2. Photosynthesis and calcification during the population changes of P. carterae in photobioreactor The population growth curve of P. carterae in photobioreactor was shown in Fig. 6. It was observed that P. carterae grew quickly during the first 3 days, with the maximum specific growth rate per day of 0.66 at day 3. The maximum cell density was increased to be 29  104 cells/mL at day 4. Then the cells became to aggregate which made the swimming cell density decrease under microscopic counting. Simultaneous measurement of photosynthetic oxygen release and cell surface calcification indicated that the inverse correlation between those two indexes still existed (Fig. 7). That was to say, with the increase of the content of CaCO3 on the cell surface, the photosynthetic oxygen release rate was decreased, and vice versa. This correlation made the both curves of photosynthetic oxygen release and cell surface calcification fluctuated. It was an intriguing phenomenon for coccolithophorids. 4. Discussions

Ca concentration (mg.L )

0

40

12 h ΔF

0.3

0

60

2.5

0.0

concentration (mg.L )

0-12 h Photosynthetic oxygen evolution

(×10-7µmol.cell -1)

3.5

Crystal CaCO3

Photosynthetic O 2 evolution (µ mol O 2 /(mg Chla•h))

1 0.5

Photosynthetic O2 evolution (µmol O 2/(mg Chla•h))

41

C. Zhou et al. / Acta Ecologica Sinica 32 (2012) 38–43

5

6

Time (days) Fig. 6. Growth of P. carterae in a batch culture in photobioreactor. Error bars = +/ 1 SD and n = 3.

lar calcium concentration was increased with the extracellular calcium levels, and the re-calcification rate was increased more rapidly than the intracellular calcium level, which can be observed by comparing the curve shape between Figs. 4 and 5. The photosynthetic

The contribution of carbon dioxide in the ocean by coccolithophorids blooms has been involved in both photosynthesis and calcification. Data available from the literature indicated that the carbon assimilation ration between calcification and photosynthesis (C/P) was ranged from 0.5 to 2.3 [21]. The critical value to determine whether coccolithophorids absorb or release CO2 is 1.5 [22]. However, more intensive studies are much needed to assess their effects on the global climate changes. Calcium as the calcification substrate of coccolithophorids plays a very important role for the formation of coccoliths crystal layers. Blackwelder et al. [23] pointed that the re-calcification of N-cells of P. carterae can be achieved 75% during 24 h with calcium concentration higher than 102 M, achieved 100% in 2 days, but failed with calcium concentration at 103 or 104 M. Katagiri et al. [24] reported that calcium concentration can affect the formation of coccoliths for Pleurochrysis haptonemofera, showing positive effects on calcification rate with calcium concentration ranged from 0 to 10 mM. However, cell physiological activity was inhibited when [Ca2+] > 50 mM. Our studies confirmed such effects were existed for a strain of P. carterae collected from eastern China Sea coastal area. The intracellular calcium content and calcification rate were both positively correlated with extracellular calcium concentration ranged from 0 to 100 mg/L (0–2.5 mM).

42

C. Zhou et al. / Acta Ecologica Sinica 32 (2012) 38–43

The relationship between photosynthesis and calcification in coccolithophorids was still largely inconclusive. Studies on E. huxleyi, which is the most intensively studies species for coccolithophorids, indicated that photosynthesis was dependent on calcification, and calcification can promote photosynthesis. One explanation is that the carbon dioxide released from calcification can be used as a readily available source for chloroplast, to increase the dissolution of carbon dioxide inside the cell wall, therefore, provide more carbon dioxide for chloroplast [4]. On the other hand, calcification can consume some excessive energy which may damage the photosynthesis [25]. Paasche [26] found that E. huxleyi grows very well in calcium free medium, without calcification, and the N-cells have the same, or more effective, photosynthetic activities as the C-cells. Herfort [27] argued that photosynthesis is not dependent on calcification, the N-cells and C-cells did not show any significant difference on photosynthesis, one more evidence was that photosynthesis was not affected by broad-spectrum inhibitor of anion exchangers such as 4,40 -diisothiocyanatostilbene-2,20 -disulfonic acid (DIDS) and 4-acetamido-40 -isothiocyanato-stilbene-2,20 -disulfonic acid (SITS), however, the adaptation of E. huxleyi for carbon assimilation during long term culture was not excluded [28]. Leonardos et al. [29] also support that photosynthesis may be not tightly coupled to calcification. Hence, the coupling of photosynthesis with calcification should be elucidated by considering environmental changes. For other coccolithophorids species, the relationships between calcification and photosynthesis are also different, depending on specific species and culture conditions. Some references have suggested that calcification neither promote photosynthesis efficiency, nor improve CO2 concentration [30–32]. In our studies, photosynthesis is inversely coupled with calcification, on both cellular and population dynamics assays. At cellular level, addition of extracellular Ca2+ increase the calcification rate with higher crystal carbonate content, but inhibit photosynthetic oxygen release, especially when the concentration of Ca2+ is above 50 mg/L. At population dynamics, the fluctuation of photosynthetic oxygen release is closely inversed with the content of carbonate content on the cell surface. The relationship between photosynthesis and calcification in coccolithophorids should be considered by taking account of extracellular carbon. If bicarbonate HCO 3 is the same source for both photosynthesis and calcification, then calcification can improve photosynthetic efficiency. However, if CO2 can permeate the cell membrane and directly absorbed, calcification could be independent on photosynthesis [7]. Studies by 14C tracing technique showed that the end products for 14CO2 is soluble organic compounds, and H14 CO 3 produce CaCO3 and insoluble organic compounds, and deduced that the major source for calcification in E. huxleyi is HCO 3 and CO2 for photosynthesis [33]. Israel and González [7] found that CO2 is the only source for photosynthesis, whereas HCO 3 and CO2 are used in calcification process for high and low calcification strains of Pleurochrysis sp. respectively. The calcification process of coccolithophorids is highly affected by cellular physiology, as well as illumination, temperature, and environmental nutrients. The new global ocean acidification can 2 greatly change the chemical composition of CO2, HCO 3 , and CO3 , which can inevitably affect the photosynthesis and calcification of coccolithophorids. Therefore, to know more about the role of coccolithophorids in ocean ecology and carbon biogeochemical global cycle, the detailed studies on coccolithophorids should be considering different species and different biogeography. Acknowledgments This work was supported by grants from NSFC Project (40776064); Ningbo Science and Technology Project (2007A31

004); Natural Science Foundation of Zhejiang (Z3100565); Project of Ministry of Education (200816460002); K.C. Wong Magna Fund in Ningbo University; Program for Changjiang Scholars, Innovative Research Team in University (No. IRT0734). References [1] P. Westbroek, J.R. Young, K. Linschooten, Coccolith production (biomineralization) in the marine alga Emiliania huxleyi, Journal of Eukaryotic Microbiology 36 (4) (1989) 368–373. [2] M.E. Marsh, Regulation of CaCO3 formation in coccolithophores, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 136 (4) (2003) 743–754. [3] D. Ariovich, R.N. Pienaar, The role of light in the incorporation and utilization of Ca2+ ions by Hymenomonas carterae (Braarud et Fagerl.) Braarud (Prymnesiophyceae), European Journal of Phycology 14 (1) (1979) 17–24. [4] S.C. Steven, R.D. Roer, K.M. Wilbur, Photosynthesis and coccolith formation: inorganic carbon sources and net inorganic reaction of deposition, Limnology and Oceanography 25 (2) (1980) 248–261. [5] T.A. McConnaughey, J.F. Whelan, Calcification generates protons for nutrient and bicarbonate uptake, Earth-Science Reviews 42 (1–2) (1997) 95–117. [6] W.M. Balch, P.M. Holligan, K.A. Kilpatrick, Calcification, photosynthesis and growth in the bloom-forming coccolithophore, Emiliania huxleyi, Continental Shelf Research 12 (12) (1992) 1353–1374. [7] A.A. Israel, E.L. González, Photosynthesis and inorganic carbon utilization in Pleurochrysis sp. (Haptophyta), a coccolithophorid alga, Marine Ecology Progress Series 137 (1996) 243–250. [8] G. Langer, M. Geisen, K.H. Baumann, J. Kläs, U. Riebesell, S. Thoms, J.R. Young, Species-specific responses of calcifying algae to changing seawater carbonate chemistry, Geochemistry Geophysics Geosystems 7 (2006), doi:10.1029/ 2005GC001227. Q09006. [9] U. Riebesell, I. Zondervan, B. Rost, P.D. Tortell, R.E. Zeebe, F.M.M. Morel, Reduced calcification of marine plankton in response to increased atmospheric CO2, Nature 407 (2000) 364–367. [10] I. Zondervan, R.E. Zeebe, B. Rost, U. Riebesell, Decreasing marine biogenic calcification: a negative feedback on rising atmospheric pCO2, Global Biogeochemical Cycles 15 (2) (2001) 507–516. [11] M.D. Iglesias-Rodriguez, P.R. Halloran, R.E.M. Rickaby, I.R. Hall, E. ColmeneroHidalgo, J.R. Gittins, D.R.H. Green, T. Tyrrell, S.J. Gibbs, P. von Dassow, E. Rehm, E.V. Armbrust, K.P. Boessenkool, Phytoplankton calcification in a high-CO2 world, Science 320 (5874) (2008) 336–340. [12] B.E. Casareto, M.P. Niraula, H. Fujimura, Y. Suzuki, Effects of carbon dioxide on the coccolithophorid Pleurochrysis carterae in incubation experiments, Aquatic Biology 7 (1–2) (2009) 59–70. [13] J. Horita, H. Zimmermann, H.D. Holland, Chemical evolution of seawater during the phanerozoic: implications from the record of marine evaporates, Geochimica et Cosmochimica Acta 66 (21) (2002) 3733–3756. [14] C.X. Zhou, B. Ma, F.X. Wang, B. Xu, X.J. Yan, The population growth and variation of nitrate-N and phosphate-P in the mix-culture of phaeodactylum tricornutum bohl and prorocentrum MICANS, Marine Sciences 30 (12) (2006) 58–61. [15] P.J. Harrison, R.E. Waters, F.J.R. Taylor, A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton, Journal of Phycology 16 (1) (1980) 28–35. [16] X. Huo, Y.Y. Bao, H. Huang, X.X. Huang, Y.J. Piao, Free Ca2+ measurement in cultured macrophages of chick embryos by laser scanning confocal microscopy with Fluo-3/ AM, Laser Journal 19 (3) (1998) 35–37. [17] D.F. Zhu, L.P. Shen, J.M. Shen, Q. Wu, Changes in intracellular Ca2+ of sperm fore-and-aft acrosome reaction in the swimming crab Portunus trituberculatus, Acta Zoologica Sinica 54 (5) (2008) 885–889. [18] S.W. Jefferey, G.F. Humphrey, New spectrophotometric equations for determine chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton, Biochem Physiol Pflanzen 167 (2) (1975) 191–194. [19] Z.F. Wang, B.C. Song, J. Zhang, Precise measurement of Ca and P content in hydroxyapatite by chemical analysis, Bulletin of the Chinese Ceramic Society 26 (1) (2007) 186–189. [20] R.Q. Du, Biostatistics, 2nd Ed., Higher Education Press, Beijing, 2003. pp. 105– 114. [21] E. Paasche, A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation, and calcification-photosynthesis interactions, Phycologia 40 (6) (2001) 503–529. [22] F. Michel, C. Canon, J.P. Gattuso, Marine calcification as a source of carbon dioxide: positive feedback of increasing atmospheric CO2, Limnology and Oceanography 39 (2) (1994) 458–462. [23] P.L. Blackwelder, R.E. Weiss, K.M. Wilbur, Effects of calcium strontium and magnesium on the coccolithophorid Cricosphaera (Hymenomonas) carterae, I Calcification Marine Biology 34 (1) (1976) 11–16. [24] F. Katagiri, Y. Takatsuka, S. Fujiwara, M. Tsuzuki, Effects of Ca and Mg on growth and calcification of the coccolithophorid Pleurochrysis haptonemofera: Ca requirement for cell division in coccolith-bearing cells and for normal coccolith formation with acidic polysaccharides, Marine Biotechnology 12 (1) (2010) 42–51. [25] B. Delille, J. Harlay, I. Zondervan, S. Jacquet, L. Chou, R. Wollast, R.G.J. Bellerby, M. Frankignoulle, A.V. Borges, U. Riebesell, J.P. Gattuso, Response of primary

C. Zhou et al. / Acta Ecologica Sinica 32 (2012) 38–43

[26]

[27] [28]

[29]

production and calcification to changes of pCO2 during experimental blooms of the coccolithophorid Emiliania huxleyi, Global Biogeochemical Cycles 19 (2005), doi:10.1029/2004GB002318. GB2023. E. Paasche, A tracer study of the inorganic carbon uptake during coccolith formation and photosynthesis in the coccolithophorid Coccolithus huxleyi, Physiolofia Plantarum Supplement 3 (1964) 1–82. L. Herfort, B. Thake, J. Roberts, Acquisition and use of bicarbonate by Emiliania huxleyi, New Phytologist 156 (3) (2002) 427–436. C. Brownlee, A. Taylor, Calcification in Coccolithophores: a Cellular Perspective, in: H.R. Theirstein, J.R. Young (Eds.), Coccolithophores: From Molecular Processes to Global Impact, Springer-Verlag Berlin Heidelberg, Berlin, 2004. pp. 31–50. N. Leonardos, B. Read, B. Thake, J.R. Young, No mechanistic dependence of photosynthesis on calcification in the coccolithophorid Emiliania Huxleyi (Haptophyta), Journal of Phycology 45 (5) (2009) 1046–1051.

43

[30] R. Björn, U. Riebesell, S. Burrkhardt, D. Sültemeyer, Carbon acquisition of bloom-forming marine phytoplankton, Limnology and Oceanography 48 (1) (2003) 55–67. [31] L. Berry, A.R. Taylor, U. Lucken, K.P. Ryan, C. Brownlee, Calcification and inorganic carbon acquisition in coccolithophores, Functional Plant Biology 29 (3) (2002) 289–299. [32] I. Zondervan, B. Rost, U. Riebesell, Effect of CO2 concentration on the PIC/POC ratio in the coccolithophore Emiliania huxleyi grown under light-limiting conditions and different day lengths, Journal of Experimental Marine Biology and Ecology 272 (1) (2002) 55–70. [33] K. Sekino, Y. Shiraiwa, Accumulation and utilization of dissolved inorganic carbon by a marine unicellular coccolithophorid Emiliania huxleyi, Plant and Cell Physiology 35 (3) (1994) 353–361.