Calcification response of Pleurochrysis carterae to iron concentrations in batch incubations: implication for the marine biogeochemical cycle

Abstract

Calcified coccolithophores, a diverse and widely distributed group of marine microalgae, produce biogenic calcite in the form of coccoliths located on the cell surface. Using batch incubations of the coccolithophoridPleurochrysis carterae, we investigated the responses of this calcification process to iron concentrations by changing the iron supply in the initial culture media from a normal concentration to 1 ppm (parts per million), 5 ppm, and 10 ppm. Time-dependent measurements of cell population, production of inorganic carbon (coccoliths), and organic carbon (organic cellular components) showed that elevated iron supply in the growth medium ofP. carterae stimulates carbon sequestration by increasing growth along enhanced photosynthetic activity and calcification. In addition, the acquired time-dependent UV-Vis and FT-IR spectra revealed that iron fertilization-enhanced coccolith calcification is accompanied by a crystalline phase transition from calcite to aragonite or amorphous phase. Our results suggest that iron concentration has a significant influence on the marine carbon cycle of coccolithophores.

Keywords

calcification / coccolithophores / iron fertilization / Pleurochrysis carterae

Introduction

Iron (Fe) is an essential micronutrient used by marine phytoplankton for the biosynthesis of pigments (chlorophyll and phycobilin) and enzymes (cytochrome, catalase, and peroxidase) involved in numerous metabolic processes such as photosynthesis and the catalysis of intracellular redox reactions (Macrellis et al., 2001; Wang and Dei, 2001; Arrigo, 2005). In marine ecosystems, iron availability is an important factor limiting the primary production of carbon undertaken by the phytoplankton (Shi et al., 2010). This also affects the marine biogeochemical cycle of carbon since the activity of phytoplankton is responsible for the sequestration of atmospheric carbon dioxide to the deep sea over large areas of the ocean (Tortell et al., 1999; Bowie et al., 2001; Hassler et al., 2015). Therefore, the influence of iron on marine phytoplankton has received growing interest from biogeochemists over the past three decades (Tortell et al., 1999; Hassler et al., 2015). Since 1993, many scientists have completed iron enrichment experiments, ranging from bottle incubations to large-scale (50‒100 km²) open ocean regimes, and have unequivocally demonstrated that iron fertilization stimulates primary production and exerts controls on the dynamics of plankton blooms (Bowie et al., 2001; Blain et al., 2007; Boyd and Ellwood, 2010). Coccolithophores represent a group of unicellular, eukaryotic phytoplankton (algae) constituted by a wide range of species, many of which are enveloped by biogenic calcite scales forming a spherical armor called coccosphere (Young et al., 1999; Müller et al., 2008). Coccolithophores contribute to the oceanic carbon cycle by conducting photosynthesis and calcification, during which dissolved inorganic carbon is used to form organic cellular components and calcium carbonates (coccoliths), respectively, thereby causing a net drawdown of CO₂ removal from the atmosphere into the ocean (Rost and Riebesell, 2004; Sun et al., 2014; Jin et al., 2015). Although iron also influences the productivity of coccolithophores, which in turn affects the biogeochemical cycle of carbon, relatively little attention has been given to this particular group. Furthermore, the influence of iron on calcification of coccoliths may be recorded in coccoliths, which can be used as an indicator of environmental change. Therefore, we used the experimental model species of coccolithophoresPleurochrysis carterae to investigate iron modulated growth, with particular attention to the aspect of calcification during batch incubation experiments by changing iron concentrations in the initial culturing medium using a FeCl₃ solution (Xu et al., 2011; Chow et al., 2015; Xing et al., 2015).

Materials and methods

Species and culture condition

P. carterae strains used in this study were maintained in an Aquil artificial seawater medium (with the following major ions content: Ca²⁺ 420 ppm (parts per million), Mg²⁺ 1310 ppm, Sr²⁺ 5.6 ppm, Fe³⁺ 0.06 ppm, and EDTA 5 ppm) (Xing et al., 2015; Xu and Gao, 2015), and were obtained from Xiamen University. The cell cultures were incubated in a light incubator (MGC-450HPY-2, Yiheng, Shanghai, China) at a constant temperature of 20°C, under a light exposure of 20,000 lux, and with photoperiod cycles of 12 h:12 h (light:dark). At least 10 generations of cells were grown before starting the iron enrichment experiments. The initial cell concentration was 6×10⁵ cells·mL^‒1.

All experiments in this study were carried out using a batch culturing method in triplicate at each iron concentration. Values are the means of triplicate cultures at each treatment±standard deviation (SD). Iron concentrations in the initial culturing medium were adjusted using a 0.2 mol/L FeCl₃ solution from standard medium concentration of 0.06 ppm, to 1 ppm, 5 ppm, and 10 ppm.

Determination of cell populations and specific growth rates

Using a blood cell counting chamber under a light microscope, each sample of the cell population was measured three times to determine the mean values and SD. Mean values were then used to draw growth curves. The specific growth rate (µ) was calculated as µ= (lnc₁‒lnc₀)/(t₁‒t₀), where c₀ and c₁ are cell concentrations at the time intervals t₀ and t₁ (t₁‒t₀=24 or 12 h) (Xing et al., 2015).

Calculations of physiological parameters

In order to measure the particulate inorganic carbon (PIC) and particulate organic carbon (POC) contents, 15 mL were collected from each cultured population. Both PIC and POC were quantified by a total carbon analyzer (LIQUID TOCII, Germany). They were calculated using the difference between the values measured in the growth medium, which contained the cells, and in the supernatant after the cells were removed using a centrifuge at 8000 rpm for 10 min. PIC and POC were thus calculated as follows: PIC= TIC‒DIC, POC= TOC‒DOC, where TIC includes both the inorganic carbon of the PIC (coccoliths) and the dissolved inorganic carbon (DIC) in the growth medium (without the coccoliths), and where TOC includes both the organic carbon of the POC (cells) and the dissolved organic carbon (DOC) in the growth medium (without the cells). The PIC and the POC production per cell were calculated according to P_PIC= (PIC/cell)×µ and P_POC=(POC/cell)×µ, respectively (Guan and Gao, 2010).

Determinations of elemental ratios

Samples for elemental quantifications were collected at the different testing points (culturing time) by centrifuging them in 15 mL Falcon tubes at 8000 rpm for 10 min. The cell pellets were transferred into 1.5 mL microcentrifuge tubes. To remove the organic matter, samples were boiled in 1 mL of a 10% NaOCl solution for 2 h. After thorough rinsing with ultrapure water, the washing procedures were repeated 3 times. The cleaned samples of coccoliths were recovered in 2% HNO₃ and put in an ultrasonic bath for 5 min to ensure complete dissolution of all the carbonate coccoliths present.

All elemental quantifications of Ca, Mg, and Sr in coccoliths were carried out by Inductively Coupled Plasma-Atomic Emission spectroscopy (ICP-AES, Icap6000, Thermo, America) (Rickaby et al., 2002). The resulting precision is higher than 0.5% on the basis that analyses of the same sample were replicated at different dilutions over the course of several days. The calcification rate (P_Ca) was calculated according to P_Ca =µ×calcite/cell, where the calcite content represented the concentration of Ca measured in the coccoliths (Langer et al., 2006). The above values were determined from the mean of three samples±SD.

Fourier Transform Infrared Spectroscopy (FT-IR) and Ultraviolet-visible spectroscopy (UV-Vis)

Each 50 mL culture was filtered using Whatman GF/F quartz fiber filters (25 mm) to record FTIR and reflecting light spectra through UV-Vis bands. Spectra of quartz filters with coccoliths were measured by a Fourier Transform Infrared Spectrometer (ATR-FTIR, FT-IR5700, Thermo, America) and an Ultraviolet-visible spectrophotometer (UV-Vis, UV-3150, Shimadzu Corporation, Japan).

Statistical analyses

The experimental data were analyzed by Statistical Product and Service Solutions (SPSS). A t-test and single factor analysis of variance (One-way ANOVA) were used for significant difference analysis. The significance level for all of the tests was set atP=0.05. Data is reported as mean±SD.

Results

The growth dynamics of P. carterae populations were calculated during incubation experiments of iron fertilization. The obtained growth curves ofP. carterae under various batch culturing conditions are shown in Fig. 1. The curves present typical inverted ‘U’ exponential growth patterns (Fig. 1(a)). After a lag phase of one day, cells grew exponentially from day 2 to day 4, with the highest growth rates of 0.02, 0.021, 0.023, and 0.026 per day in the standard, Fe: 1 ppm, Fe: 5 ppm, and Fe: 10 ppm culture mediums, respectively.

The relative efficiency of the two biological carbon fixation processes, namely photosynthesis and calcification, are given by the ratio of PIC/POC, which reflects the flux of CO₂ between the surface ocean and the overlying atmosphere (Rost and Riebesell, 2004). Our results show that PIC productivity decreases significantly over time for each of the batch culturing experiments (Fig. 2(a)), while POC productivity is higher in the case of iron enrichment culturing conditions compared to the normal culturing condition (Fig. 2(b)). A decreasing trend of the PIC/POC ratio was observed with increasing iron supply (Fig. 2(c)). More specifically, at the time-length of 96 h of batch cultures, the PIC/POC ratios dropped to 42%, 44%, and 33%, in response to 1 ppm, 5 ppm, and 10 ppm content of iron in culture mediums, respectively.

Since it was shown that Sr/Ca and Mg/Ca ratios in coccoliths of P. carterae provide important implications for the biogeochemical cycles (Langer et al., 2006; O’Dea et al., 2014), we calculated these ratios under our iron enrichment experiments (Fig. 3). Results show a significant variability of Sr/Ca and Mg/Ca with increasing iron content (Fig. 3). The ratios of Sr/Ca were decreased with increasing iron concentrations indicating that the calcification was enhanced for increasing Ca contents. However, the ratios of Mg/Ca were increased with increasing iron concentrations showing that Mg was co-incorporated into the calcium carbonate of coccoliths ofP. carterae during enhanced calcifications.

In the region of a UV light band (200‒300 nm), the absorption of P. carterae coccoliths was negligible (Fig. 4). However, the absorption reached its maximum value in the band of 300‒500 nm, overlapping the absorption band of iron (300‒580 nm).

Since FT-IR spectroscopy is a powerful tool for studying polymorphs of calcium carbonate, we recorded FT-IR spectra ofP. carterae during batch culturing in the range of 2600 to 600 cm^‒1. Previous studies showed that FT-IR spectra of calcium carbonate containing symmetric stretching vibration (ѵ1 mode), antisymmetric stretching vibration (ѵ2 mode), out-of-plane bending (ѵ3 mode), and in-plane bending vibration (ѵ4 mode), appeared at 1083 cm^‒1, 1433 cm^‒1, 878 cm^‒1, and 713 cm^‒1, respectively (Li et al., 2013; Wang et al., 2015). The bands at 1646 cm^‒1 (Fig. 5) can be attributed to uronic acid, which is an organic ligand secreted by coccolithophores. Meanwhile, the bands at 2337 cm^‒1 and 2358 cm^‒1 (Fig. 5(b)) can be attributed to O-C-O switching. The characteristic bands at 1020 cm^‒1 in the FT-IR spectra of normal culturing correspond to the calcite from the coccoliths (Fig. 5(a)). When a specific amount of iron was added into the culture media, red shifting occurred in the characteristic bands of coccolithophores at 996 cm^‒1, implying that iron influenced the mineral phase transition during the iron-associated biomineralization process. Moreover, the bands at 996 cm^‒1 and 998 cm^‒1 can be attributed to amorphous calcium carbonate in a culture medium with iron concentrations at 1 ppm and 5 ppm (Figs. 5(b) and 5(c)). The band at 1002 cm^‒1 in the case of an iron supply of 10 ppm should correspond to the characteristic band of aragonite (Fig. 5(d)). Therefore, our results show that iron incorporation into the cell surface or its uptake in the cytoplasm leads to a transition of the mineral phase constituting the coccoliths from calcite to aragonite or to amorphous calcium carbonate.

Discussion

The batch culturing experiments of iron concentrations have shown that the cell populations exponentially increased throughout to a maximum 2-fold of normal values ofP. carterae, which indicates that an elevated iron supply stimulates the growth of cells (Fig. 1). The PIC productivity was increased due to the formation of biogenic coccoliths (CaCO₃) by calcification during cell growth (Fig. 2(a)), while an increase in POC productivity is noted due to the photosynthetic process (Fig. 2(b)). Therefore, our results show that the addition of iron stimulated the growth ofP. carterae while enhancing both the photosynthesis activity and the calcification process. This in turn affects carbon fixation by increasing PIC and POC productivities.

It was shown that iron associated with exopolymeric substances (EPS) is highly bioavailable to the oceanic phytoplankton (Hassler et al., 2015). Coccolithophores can produce EPS, composed of hyaluronic acid, polysaccharide, amino acid, and proteins (Henriksen and Stipp, 2009; Chow et al., 2015). The characteristic peak of the uronic acid of EPS, the major contributor for iron-binding ligands, was detected by our FT-IR analyses (Fig. 5). Furthermore, elemental analyses of the coccoliths show a significant variability of Sr/Ca and Mg/Ca ratios with increasing iron content (Fig. 3). Coccoliths (calcite) secreted byP. carterae have a vital function: reflecting UV light while absorbing visible light, the latter being required to stimulate the metabolic activities of photosynthesis and calcification (Young et al., 2009). UV-Vis spectra showed a strong absorption peak at 680 nm, suggesting that iron enrichment enhances the absorption of visible light and the efficiency of its utilization (Fig. 4). Furthermore, we showed that the influence of iron concentrations is particularly significant on calcification. Indeed, the addition of iron altered the crystalline structure of the coccoliths (calcite) and simultaneously induced a phase transition from calcite to aragonite or to amorphous calcium carbonate.

Iron fertilization is the introduction of iron to the upper ocean to stimulate a phytoplankton bloom to increase carbon dioxide removal from the atmosphere. Our results showed that iron fertilization may also stimulate coccolithophores bloom which can influence the marine food chain and carbon cycle. Eutrophication was normally considered as the major cause of coccolithophore blooms. However, extensive blooms formed in nutrient depleted conditions. Thus, it is essential that the environmental supply of trace and micro elements be given further consideration in future studies on coccolithophore blooms.

Conclusions

Iron concentrations play an important role in the biogeochemical carbon cycle of coccolithophores in batch incubations. Elevated iron supply stimulates the growth of cells through photosynthesis and calcification, thus enhancing the efficiency of carbon fixation from dissolved inorganic carbon from the atmosphere. The results of FT-IR and UV-Vis spectra indicate that the iron modulates the calcification process of coccoliths by inducing a crystalline phase transformation from calcite to aragonite or amorphous calcium carbonate. Furthermore, our study also confirmed that iron supply has a significant influence on the Sr/Ca and Mg/Ca ratios, which provides useful information on the role of coccolithophores in the marine biogeochemical cycle. Although this study focuses on iron concentrations, we believe that coccolithophores are likely to play an important role in the oceanic cycling of trace metals as well.

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