The centric diatom T. pseudonana (Strain CS-20) was obtained from the Australian national algae culture collection in Hobart, Australia and maintained in f/2 medium (CSIRO recipe) at 20°C under a continuous photon flux density (PFD) of 120–145 μE m⁻² s⁻¹, before being transferred to Fe-replete Aquil medium (Price et al., 1988). Stock cultures of the Southern Ocean diatoms P. inermis and E. Antarctica were isolated from waters south of the Antarctic Polar Frontal Zone in December 2001 (Strzepek et al., 2011). The Southern Ocean diatoms have since been maintained in Fe-replete Aquil medium at a temperature of 3°C under a continuous photon flux density (PFD) of ~35 μE m⁻² s⁻¹. Cultures were acclimated to the different Fe concentrations in 28 mL polycarbonate vials for a minimum of three transfers (approximately 10–12 generations), before being transferred to 1 L polycarbonate bottles for experimental work. All cultures were grown in batches rather than under semi-continuous conditions to minimize contamination from trace metals. Average PFD's for cultures of E. Antarctica, P. inermis, and T. pseudonana were 45 μE m⁻² s⁻¹, 60 μE m⁻² s⁻¹ and 133μE m⁻² s⁻¹ respectively (Sunda and Huntsman, 1997; Strzepek et al., 2012). Experimental temperatures were 20°C for T. pseudonana and 3°C for the Southern Ocean species.

Aquil medium was prepared using trace-metal ultra-clean techniques and enriched with the following nutrients; 10 μmol L⁻¹ phosphate, 100 μmol L⁻¹Si(OH)₄, 300 μmol L⁻¹ nitrate, 0.55 μg L⁻¹ vitamin B₁₂, 0.5 μg L⁻¹ Biotin and 100 μg L⁻¹thiamin. Basal medium and stock solutions were eluted through a column containing Toyopearl AF-chelate-650M (Tosohbioscience) ion-exchange resin to remove metal contaminants and filter sterilized (0.2 μm) (Price et al., 1988). Iron contamination of the basal medium was measured using high-resolution IC-PMS after pre-concentration (Ellwood et al., 2008) and found to be 0.56 ± 0.02 nmol L⁻¹ (n = 3). Trace metal ion concentrations were controlled through the addition of a trace-metal ion buffer system, which used 10 and 100 μmol L⁻¹ ethylene-diamine-tetra-acetic-acid (EDTA), as the chelating agent, for both Southern Ocean species and T. pseudonana (respectively). Trace metal concentrations were 7.91 nmol L⁻¹ ZnSO₄, 1.98 nmol L⁻¹ CuSO₄, 5 nmol L⁻¹ CoCl₂, 22.8 nmol L⁻¹ MnCl₂, 9.96 nmol L⁻¹ Na₂SeO₃ and 100 nmol L⁻¹ Na₂MoO₄ for media containing P. inermis and E. Antarctica; and 100 nmol L⁻¹ ZnSO₄, 40 nmol L⁻¹ CuSO₄, 40 nmol L⁻¹ CoCl₂, 100 nmol L⁻¹ MnCl₂, 10 nmol L⁻¹ Na₂SeO₃ and 100 nmol L⁻¹ Na₂MoO₄ for media containing T. pseudonana. Free ion concentrations were calculated using Visual MINTEQ, giving concentrations (expressed as –log free metal ion concentration = pMetal) of, pCu 14.07, pMn 8.18, pZn 10.78 and pCo 11.09 for Aquil medium at a temperature of 3°C and a pH of 8.4 for Southern Ocean media, while pMetal values for T. pseudonana media were pCu 13.65, pMn 8.35, pZn 10.72 and pCo 11.26 for Aquil medium at a temperature of 20°C and a pH of 8.1.

Culture pH was measured by the change in absorbance of m-cresol purple in culture medium at specific wavelengths of 434, 578, and 730 nm respectively on a Varian Cary 1E UV-visible spectrophotometer with attached temperature controller (Clayton and Byrne, 1993; Yao et al., 2007). Measurements were made at 25°C and corrected for dye-induced changes in the pH. The pH was then adjusted to the culture temperature of 20°C using CO2SYS (http://cdiac.ornl.gov/oceans/co2rprt.html).

T. pseudonana cultures were treated with a range of Fe concentrations varying from Fe limiting to Fe replete: from 30 through to 50, 80, 250, and 500 nmol L⁻¹ of total dissolved Fe, equating to –log Fe³⁺ concentration (pFe) values of 20.59, 20.36, 20.16, 19.67, and 19.36 respectively. The Fe concentrations were selected to induce different degrees of limitation as defined by the reduction in growth rate from μmax. Inorganic Fe concentrations (Fe′) were calculated according to Sunda and Huntsman (2003) for a temperature of 20°C, a mean irradiance of 133 μE m⁻² s⁻¹ and a mean starting pH of 7.98. Fe′ values based on total dissolved Fe concentrations of 500, 250, 80, 50, and 30 nM equated to 0.42, 0.21, 0.07, 0.04, and 0.02 nM respectively (Table 1) (Sunda and Huntsman, 2003).

Southern Ocean cultures were treated differently to T. pseudonana cultures, and two competing ligands were used to induce Fe-limitation. This is because Southern Ocean diatoms are well adapted to iron limiting conditions over coastal isolates such as T. pseudonana. Iron replete media was prepared through the addition of a filter-sterilized FeEDTA complex (1:1.05[mol:mol]) to Aquil containing 10 μmol L⁻¹ EDTA for a final concentration (including contamination) of 58.3 nmol L⁻¹ of total dissolved Fe. Iron limited media was prepared by adding Fe pre-complexed with the terrestrial siderophore desferrioxamine B mesylate (DFB; Sigma Aldridge) to Aquil media containing 10 μMol L⁻¹ EDTA for a final concentration of 4.4 nmol L⁻¹. Varying degrees of Fe limitation were induced in culture by increasing the amount of DFB in culture at concentrations of 40 and 80 nmol L⁻¹ for E. Antarctica and P. inermis (respectively). The Fe′ concentration in the iron-replete media was calculated according to Sunda and Huntsman (2003) for a temperature of 3°C, a mean irradiance of 52 μE m⁻² s⁻¹ and a pH of 8.4. The overall conditional dissociation constant (Kd′) was calculated according to methods described by Strzepek et al. (2011) and represents the sum of the conditional stability constant in the dark (3.52 × 10⁻⁷) and the conditional photo-dissociation constant (K_hv = 2.17 × 10⁻⁶) of EDTA at 3°C. The Fe′ concentration for FeDFB treatment was calculated according to the equation [Fe′] = [FeDFB] / [L′] × KFe′Lcond; where KFe′Lcond = 10^11.8 (Maldonado et al., 2005). In the instance where total Fe exceeded the concentration of DFB bound Fe (Fe_tot = 4.4 nmol L⁻¹; DFB = 4 nmol L⁻¹), we assumed a 0.1 nmol L⁻¹ excess of DFB in the media and a 0.5 nmol L⁻¹ excess of total Fe that was available for interaction with EDTA (Table 1).

Once cultures were in their exponential phase of growth, 50–100 ml of cell culture was collected onto a 2 μm, 25 mm polycarbonate filter, rinsed with nutrient free Aquil, and washed into 3 mL Teflon bombs. Purification of the samples were done according to similar methods for sponge spicules by Wille et al. (2010). The samples were evaporated to dryness at 50°C in a drying oven overnight. To remove organics, samples were treated with 1 mL of 30% hydrogen-peroxide (H₂O₂) solution, and allowed to reflux for 24 h at 70°C on a hotplate. Following oxidation, the lids were removed, and the samples were left to evaporate to dryness before adding 2 ml of 0.5 M sodium hydroxide (NaOH) to dissolve the sediment. Samples were then left to reflux overnight again for 24 h at 50°C. The Si(OH)₄concentration within the sample digest was then measured colorimetrically (Strickland and Parsons, 1965). Sodium was removed from the samples using cation exchange columns to prevent any subsequent interference during isotope determination. Cation exchange columns consisted on 1 mL Dowex 50W-X8 cation exchange resin (200–400 mesh) with a 2.5 mL reservoir. Columns were cleaned with 0.5 mL 8% (w/w) hydrofluoric acid (HF) followed by 2.25 mL of deionized water. The resin with protonated by passing 2.25 mL of 4 mol L⁻¹ HCl followed by 2.25 mL of deionized water. The columns were then loaded with 0.5 mL of sample, before being rinsed with 4 × 0.5 mL of deionized water. To minimize contamination, samples were collected in vials that were cleaned using 8% (w/w) HF, before being rinsed with deionized water. Samples for δ³⁰Si determination of the growth media were prepared by dissolving the sodium silicate standard used for nutrient enrichment in the growth medium to a concentration of 2 mmol L⁻¹ in separate solutions of nutrient free Aquil and 0.5 M sodium hydroxide. These samples were then treated with the same column procedure as the cell samples prior to analysis.

The δ³⁰Si composition of diatom silica was determined according to methods developed by Wille et al. (2010) using a multi-collector inductively coupled plasma mass spectrometer (MC-ICP-MS) (Finnigan Neptune, Germany) operating in dry plasma mode at medium-resolution (M/ΔM ~2,000). An ESI-Apex nebulizer fitted with a Teflon inlet system and a demountable torch fitted with an alumina injector was used for sample introduction to minimize any background interference. A standard-sample-standard bracketing technique was used for data acquisition and reduction (Wille et al., 2010). The δ³⁰Si signal (based on the relative abundance of ³⁰Si to ²⁸Si (³⁰Si/²⁸Si) was calculated using the following formula:

Where R_sample is the ratio of ³⁰Si/²⁸ Si of the sample and R_Std is the ³⁰Si/²⁸Si of the in-house standard developed from dissolution a diatomaceous sediment and purified (Figure 1). Measurements of sample blanks were made prior to each run to ensure that the combined blank and background was <1% of the total sample signal. Inter-laboratory NBS-28 and diatomite standards were prepared with each daily run, and were measured with every three samples (≤ 8 diatomite/NBS -28 standards per daily run).

δ³⁰Si and δ²⁹Si values relative to NBS-28 plotted on an mass-dependent fractionation line with a slope of 0.520 ± 0.009 and is consistent with the consensus slope of 0.511 obtained from inter-laboratory silicon standard measurements (Reynolds et al., 2007). The reproducibility of the δ³⁰Si signal measured for the NBS-28 standard (prepared in full and measured on 5 separate occasions) was 0.25 ± 0.04 ‰. Measurements of the “diatomite standard” produced a mean δ³⁰Si value of 1.29 ± 0.25 ‰ (2 SD, n = 33), and is in good agreement with inter-laboratory comparisons (δ³⁰Si = 1.27 ‰) (Reynolds et al., 2007).

T. pseudonana displayed little variation in ε with decreasing Fe concentration (Figure 2). No clear trends were observed across the five Fe-conditions, with ε values ranging between 0.51 ± 0.46 ‰ and 0.75 ± 0.24 ‰. Comparisons of ε values between Fe-limited (Fe′ = 208, 67, 42, and 25 pmol L⁻¹) and Fe-replete conditions (Fe′ = 418 pmol L⁻¹) were not statistically significant (ANOVA, p > 0.1). Because of the statistical insignificance between these ε values, the mean was taken of all values (−0.61 ± 0.21 ‰) and compared against ε values obtained from two strains of T. pseudonana (CCMP1014 and CCCM58, −0.97 ± 0.14 and −0.88 ± 0.06 ‰, respectively) cultured under Fe-replete conditions by Sutton et al. (2013). We observed a difference (p < 0.05) between the ε in our strain (CS-20) and the two strains grown by Sutton et al. (2013) (Table 2).

Mean values for ε varied between the Fe-replete and Fe-limited conditions for the two Southern Ocean diatoms (Table 2). While there was some variability in the values of ε for E. Antarctica, there was a significant difference in ε (p = 0.04) between Fe-replete and Fe-limited conditions, with mean ε values being more negative for Fe-limited cultures (Fe-limited, ε = −1.57 ± 0.50 ‰; Fe-replete, ε = −1.40 ± 0.41 ‰) (Table 1). Mean ε values were also more negative for P. inermis cultured under Fe-limited conditions (Fe-limited, ε = −1.38 ± 0.27 ‰; Fe-replete, ε = −1.11 ± 0.15 ‰). Both Fe-replete and Fe-limited datasets for P. inermis exhibited a significant difference at the 90% confidence interval (p = 0.08), and removing outliers from both Fe-replete and Fe-limited data sets for P. inermis makes the difference more significant (p = 0.04).

Whilst little effect in the variability of the Si-isotope fractionation factor was observed in T. pseudonana in response to Fe-stress (Figure 2B), both Southern Ocean species exhibited a fractionation factor that was more negative under Fe-limitation (Figure 2A). Values for ε have been observed to vary under optimal growth conditions between diatom species (Sutton et al., 2013), so it is possible that there may be an inter-species effect when it comes to environmental controls on Si-isotope fractionation in diatoms. Sutton et al. (2013) demonstrated that Si-isotope fractionation can vary by as much as 1.5 ‰ between diatom species, enough to potentially confound palaeoceanographic interpretations utilizing δ³⁰Si from diatom opal to estimate the nutrient status of the surface ocean. In addition to interspecies effects, variations in the value for ε between individual diatom strains have been observed (Sutton et al., 2013). Silicon isotope fractionation independently measured in T. weissflogii across three seperate studies yielded ε values ranging between −0.72 ± 0.04 ‰ and −1.5 ± 0.1 ‰ (De La Rocha et al., 1997; Milligan et al., 2004; Sutton et al., 2013). Similar variations have also been observed for three T. pseudonana clones; −0.61 ± 0.21 ‰ measured in this study and −0.94 ± 0.14 ‰ and −0.88 ± 0.06 ‰ measured for two clones cultured by Sutton et al. (2013). Sutton et al. (2013) suggest that the differences in ε values for T. weissflogii can be attributable to the diatom being a coastal isolate rather than an open ocean isolate. There were remarkable similarities, however, in ε values between the two T. pseudonana strains (CCMP1014 and CCCM58) cultured by Sutton et al. (2013), even though CCMP1014 is an open ocean isolate from the (North Pacific Gyre; https://ncma.bigelow.org/) and CCCM58 is a coastal isolate (Moriches Bay, Long island, NY; http://www3.botany.ubc.ca/cccm/index.html). While it is possible that inherent differences between strains as a result of the growth conditions in their endemic environment, variations culturing conditions need to be ruled out first before this hypothesis can be invoked.

Silicon-processing in diatoms is affected by Fe bioavailability (Meyerink et al., 2017) and hence can manifest to variations in the δ³⁰Si composition of diatom silica. Understanding how diatoms fractionate Si isotopes during uptake and bio-mineralization requires a thorough investigation into how diatoms take up Si(OH)₄ from the surrounding water column and use it in the formation of their cell frustules (Sutton et al., 2013; Meyerink et al., 2017). There are numerous intracellular pathways during the synthesis of biogenic silica in diatoms where fractionation of Si isotopes can potentially occur.

In diatoms, Si(OH)₄ uptake from the surrounding water column is typically characterized either by diffusion or Michaelis-Menten saturation kinetics (Hildebrand, 2008; Javaheri et al., 2014). At low ambient Si(OH)₄ concentrations, uptake is mainly a saturatable process (≤ 10 μmol L⁻¹), and is facilitated by Si transporters (SITs) that are localized in cell membranes (Shrestha and Hildebrand, 2015). SITs are membrane bound proteins that actively take up Si(OH)₄and transport it across the outer cell membrane to the cell cytoplasm against a concentration gradient (Hildebrand, 2008). At higher ambient Si(OH)₄ concentrations (≥30 μmol L⁻¹), diffusional uptake dominates Si acquisition with SITs playing more of a regulatory role (Thamatrakoln and Hildebrand, 2008; Shrestha and Hildebrand, 2015). Passive diffusion of Si(OH)₄ appears to be proportional to the permeability of the cell membrane and the concentration gradient across the cell membrane (Hildebrand, 2008; Javaheri et al., 2014). Once past the cell membrane, Si(OH)₄ is then transported intracellularly to the Si deposition vesicle (SDV) by as yet unknown transporters/compounds classified simply as “Si binding components” (Thamatrakoln and Hildebrand, 2008). Si binding components play an important role in setting the Si(OH)₄ concentration gradient across the cell membrane under scenarios where diffusional uptake dominates (Thamatrakoln and Hildebrand, 2008). Active transport involving SITs likely plays an important role in facilitating the transport of Si from the cell cytoplasm into the SDV, as concentration gradients across the intracellular membrane separating the SDV from the cell cytoplasm are extremely high (Hildebrand, 2008). A new cell wall is created when the SDV is exocytosed at the M (mitosis) stage of the cell cycle. Cell wall formation during this stage is facilitated by silafins and long chain polyamines, which act as a “template” on to which the new cell wall is precipitated (Mock et al., 2008).

Fractionation of Si isotopes can potentially occur along any of these pathways. Milligan et al. (2004) explored where isotopic discrimination takes place within the cell by conducting a number of experiments where they varied the efflux:influx ratio of Si(OH)₄ into and out of the cell. They suggested that fractionation of Si isotopes occurs during the membrane transport step and not during Si polymerization, as Si isotope fractionation should scale linearly with the efflux:influx ratio. In addition, the relatively small Si isotope effect may also be evidence of discrimination at the transport level, as this does not necessitate the breaking or forming of chemical bonds (Milligan et al., 2004). If this is the case, fractionation of Si isotopes occurs either at the membrane/seawater interface, as Si(OH)₄ is taken up from the water column and transported to the cell cytoplasm, or, as Si is transported from the cell cytoplasm into the Silicon deposition vesicle.

Sutton et al. (2013) observed an inter-specific variation in mean ε values in Southern Ocean diatom species based on mono-culture in-vitro incubations. They argue that variability in diatom community composition could partly explain the observed variability in Southern Ocean Si-isotope fractionation factors. It is possible that variations in the Fe-status of certain regions in the Southern Ocean may contribute to variations in the apparent Si isotope fractionation factor. For example, Si isotope fractionation factors of diatom communities can potentially vary as a result of certain diatoms adapted to low Fe-conditions out competing other diatoms under Fe-limiting conditions. This may alter the δ³⁰Si_DSi composition of surface waters. While this scenario is hypothetical, it may contribute to some of the variability in the observed relationship between the mixed layer mean apparent fractionation factor (Δ³⁰Si = δ³⁰Si_DSi – δ³⁰Si_BSi) and Si(OH)₄ concentration in the Southern Ocean and the seasonal variations in Si uptake:dissolution (Varela et al., 2004; Cardinal et al., 2007; Fripiat et al., 2012).

Silicon metabolism in diatoms is particularly sensitive to variations in Fe-supply and as such, variations in ε can potentially be dependent on Fe-induced physiological variations within the cell. A notable result from our study was the relative response in the Si isotope fractionation factor (ε) of T. pseudonana under Fe-limitation compared to P. inermis and E. Antarctica. T. pseudonana exhibited no variation in ε in response to Fe-limitation, and maintained a mean ε value of –0.61 ± 0.21 ‰ across all Fe′ concentrations. In contrast, we observed a negative response in Si isotope fractionation as a result of increased Fe-limitation in both Southern Ocean species (Figure 2). The reason behind this difference could lie in how Si is metabolized by Southern Ocean diatoms under Fe-stress.

Kinetic uptake experiments show diatoms decrease their Si(OH)₄ uptake rate under Fe-limitation (Franck et al., 2000; Leynaert et al., 2004; Meyerink et al., 2017). This is generally manifested by a reduction in the maximal Si(OH)₄ uptake rate (V_Si−max) and half saturation constant (K_Si). In addition, diatoms grown under Fe-limiting conditions generally exhibit reductions in their growth rate, and variations in their cell morphology. A recent study by Meyerink et al. (2017) investigated the effects of Fe availability on the Si(OH)₄ uptake kinetics of two Southern Ocean diatoms (E. Antarctica and P. inermis) and a coastal isolate (T. pseudonana). An interesting result from the study shows that when V_Si−max is normalized to cell surface area, it exhibits a linear relationship with cell growth rate. This relationship is independent of any variations in cell morphology or species. It is likely that this relationship can be explained by the fact that silicon uptake in the diatoms was under diffusion control, as they were acclimatized under high Si conditions. Under diffusion controlled uptake, the V_Si−max values reflect the realized Si(OH)₄ uptake rates in culture which at steady state cannot exceed cellular demands for Si(OH)₄ (Thamatrakoln and Hildebrand, 2008). In contrast, variations in K_Si in response to Fe availability exhibited no relationship with other cell parameters, however it was noticeably higher in value in the Southern Ocean species than the K_Si value for T. pseudonana. This is likely because the Southern Ocean diatoms are well adapted to growing under high Si conditions.

Diatoms with a high K_Si for Si(OH)₄ such as E. Antarctica and P. inermis (≥10 μmol L⁻¹) (Meyerink et al., 2017) could possibly fractionate Si-isotopes differently to diatoms with a relatively low K_Si such as T. pseudonana because of their likely dependence on diffusional uptake of Si(OH)₄ at concentrations greater than 30 μmol L⁻¹ (Thamatrakoln and Hildebrand, 2008). Under Fe-limitation, diatoms decrease their cellular growth rate, which results in a subsequent decrease in their maximal Si(OH)₄ uptake rate (V_Si−max) (Franck et al., 2000; Leynaert et al., 2004; Meyerink et al., 2017). This may result in a decrease in a diatoms specific affinity for Si(OH)₄ that can be maintained by a decrease in the K_Si (Franck et al., 2000; Leynaert et al., 2004; Meyerink et al., 2017). Previous studies suggest that diatoms maintain their affinity by altering their cell size (Leynaert et al., 2004); however, E. Antarctica exhibits a 2-fold decrease in K_Si when Fe-limited, which is concomitant with an increase in cell-size (Meyerink et al., 2017). It is therefore more likely that diatoms maintain their affinity for Si(OH)₄ by adjusting the point where diffusive transport takes over from active transport. Active transport persists at Si(OH)₄ concentrations greater than 30 μmol L⁻¹ under Fe-limitation, and as a result, could possibly induce a decrease in the diatoms Si-isotope fractionation factor (Shrestha and Hildebrand, 2015). If this is correct, why do E. Antarctica and P. inermis exhibit a more negative response in ε when compared to T. pseudonana? It is possible that different genera of diatoms switch from active to diffusive transport at different Si(OH)₄ concentrations. While T. pseudonana does this at ~30 μmol L⁻¹ under Fe-replete conditions (Shrestha and Hildebrand, 2015), this value could be higher for Southern Ocean diatoms. When diatoms approached Fe-limiting conditions, such as in this study, T. pseudonana possibly remained under the threshold where active transport takes place, while E. Antarctica and P. inermis did not. If this is the case, then Si-isotope signatures may potentially be subject to the synergistic effects of species diversity and resource limitation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.