Investigating the effect of nickel concentration on phytoplankton growth to inform the assessment of ocean alkalinity enhancement

Ocean alkalinity enhancement (OAE) is a proposed method for removing carbon dioxide (CO2) from the atmosphere by the accelerated weathering of (ultra-) basic minerals to increase alkalinity – the chemical capacity of seawater to store CO2. During the weathering of OAE-relevant minerals relatively large amounts of trace metals will be released and may perturb pelagic ecosystems. Nickel (Ni) is of particular concern as it is 15 abundant in olivine, one of the most widely considered minerals for OAE. However, so far there is limited knowledge about the impact of Ni on marine biota including phytoplankton. To fill this knowledge gap, this study tested the growth and photo-physiological response of 11 marine phytoplankton species to a wide range of dissolved Ni concentrations (from 0.07 nmol/L to 50,000 nmol/L). We found that the phytoplankton species were not very sensitive to Ni concentrations under the culturing conditions established in our experiments, but the 20 responses were species-specific. The growth rates of 6 of the 11 tested species showed small but significant responses to changing Ni concentrations. Photosynthetic performance, assessed by measuring the maximum quantum yield (Fv/Fm) and the functional absorption cross-section (σPSII) of photosystem II, was also only mildly sensitive to changing Ni in 3 out of 11 species and 4 out of 11 species, respectively. The limited effect of Ni may be partly due to the provision of nitrate as the nitrogen source for growth, as previous studies suggest higher 25 sensitivities when urea is the nitrogen source. Furthermore, limited influence may be due to the relatively high concentrations of organic ligands in the growth media in our experiments. These ligands reduced bioavailable Ni (i.e., “free Ni”) concentrations by binding the majority of the dissolved Ni. Our data suggest that dissolved Ni https://doi.org/10.5194/bg-2021-312 Preprint. Discussion started: 3 January 2022 c © Author(s) 2022. CC BY 4.0 License.

comparability between experiments. However, this issue does not affect the interpretation of the results as all species and replicates received the same amount of light throughout the experiment. The light intensity was the average light intensity at each of the 88 spots on the phytoplankton disc measured with a Licor light meter. In vivo chlorophyll fluorescence during the growth cycle of phytoplankton cultures. We only used fluorescence values where biomass inside the polycarbonate tubes was still relatively low (maximum up to a fluorescence of 13) as indicated in this 120 example with the thick orange dots. The arrow indicates the time when the culture was usually transferred into the next batch of fresh medium. (Please note that the data illustrated here is from a test where we let the culture grow into nutrient depletion.) (c) The fluorescence values measured at low biomass were ln-transformed and plotted against time (day). The slope of the linear regression in this plot represents the specific growth rate (μ; d -1 ).

Nickel treatment
125 Aquil media were enriched with different concentrations of NiCl2: 0, 5, 10, 20, 30, 50, 70, 100, 150, 200, 300, 400, 500, 700, 1000, 10000 and 50000 nmol/L. Unless otherwise noted, "Ni concentration" refers to the total added dissolved Ni concentration. For illustration and discussion of the data, concentrations were negatively log10 transformed: where Ni is the total dissolved concentration of Ni in mol/L. This kind of transformation is also used to convert hydrogen ion concentrations to pH and is commonly used in studies investigating trace metal sensitivities to better https://doi.org/10.5194/bg-2021-312 Preprint. Discussion started: 3 January 2022 c Author(s) 2022. CC BY 4.0 License.
visualize data when trace metal concentrations vary over orders of magnitude (Dupont et al., 2008).
Media were allowed to equilibrate chemically for at least 24 h before being inoculated with phytoplankton. To acclimate the phytoplankton strains, stock cultures were first transferred into Aquil medium without Ni enrichment.

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They were then cultivated for at least 3 batch cycles (i.e., transferred from one polycarbonate tube to the next one) before being transferred to polycarbonate tubes with the different Ni treatments. This ensured that the phytoplankton species were acclimated to Aquil medium before the Ni experiment commenced.
Due to the addition of the ligand EDTA to the Aquil media, the "free Ni" ion concentrations (i.e., Ni 2+ ) were substantially lower than the total dissolved Ni concentrations as was calculated with the chemical speciation 140 software Visual MINTEQ 3.1 (Gustafsson, 2011). We were interested to see if the response of phytoplankton to Ni may be different in other growth media where no EDTA was added. Therefore, we prepared a batch of natural seawater medium with water sampled from 15 m in the Southern Ocean (58.02 °S, 141.17 °E). This natural seawater was filtered through an acid-cleaned 0.2 μm filter and sterilized in the microwave. The same amount of macro-nutrients (N, P and Si) and vitamins were added as in the Aquil medium (mentioned above). The trace metal 145 additions to the Southern Ocean seawater (no Ni included) were adjusted to a similar free trace metal concentration (nutrient-replete) as in Aquil medium (Table A1). For the experiment with natural seawater, we set up a dissolved Ni gradient with 17 concentrations: 0, 1, 2, 5, 10, 20, 30, 50, 70, 100, 150, 200, 300, 400, 500, 700, and 1000 nmol/L. The extremely high Ni concentrations designed for the Aquil medium were avoided as we assumed the organic ligand concentrations in natural seawater to be much lower than the concentration of EDTA added in Aquil 150 medium and therefore the concentration of free Ni 2+ to be higher. We used P. tricornutum (CS-29) for this experiment. Phaeodactylum tricornutum was transferred from the stock cultures into natural seawater medium for 3 batches cycles prior to the experiment with different Ni treatments as described for the Aquil medium above.

Growth rate measurement
Growth rate measurements were conducted according to the methods described by Andersen (2005). Briefly, the chlorophyll fluorescence of the cells was recorded daily at the same time with a Turner Model 10-AU fluorometer.
During the measurements, polycarbonate tubes did not have to be opened because they fit within the sample chamber of the fluorometer. This reduced the risk of contamination as the polycarbonate tubes remained closed 165 throughout the experiment. Fluorescence signals of samples were measured after 20 minutes of dark acclimation.
The fluorescence values were ln-transformed and plotted as a function of incubation days. A linear regression was fitted during the exponential phase of phytoplankton growth with the specific growth rate (μ; d -1 ) represented by the slope of the linear regression ( Fig. 1(b) and (c)). We only used fluorescence values up to 13 (arbitrary unit) for our growth rate calculations so that the biomass in the incubation bottles remained relatively low and consistent Reliable estimates of exponential growth rates in dilute batch cultures require multiple serial transfers of cultures (all performed while the strain is still in exponential growth) to allow the time for cultures to acclimate to the experimental conditions (Brand et al., 1981;Andersen, 2005). Therefore, the phytoplankton species were transferred into new polycarbonate tubes containing fresh medium during their early exponential stage for 3 batch 175 cycles prior to recording growth rates shown in the results. This meant that cultures were usually growing in their respective treatment conditions for at least three weeks.

Fast repetition rate fluorometry
We conducted photo-physiological measurements at the end of each batch cycle. (2009)), while σPSII describes the ability of light to promote a photochemical reaction in PSII (Falkowski and Raven, 195 1997). The value of Fv/Fm and σPSII are known to vary among algal taxa (Suggett et al., 2009). Typically, cells growing in batch cultures at steady state exhibit a constant value of Fv/Fm and σPSII during exponential growth phase (Parkhill et al., 2001).

Data analysis
The growth rate and photo-physiological response of phytoplankton was analysed using generalised additive in response to the wide Ni gradient (i.e., the smooth term was not significantly different from a horizontal line and therefore no statistically significant relationship between Ni and the measured parameter present). The general GAM equation is: where Y is the response variable (μ, Fv/Fm and σPSII); I0 is the intercept; S(pNi) is the non-parametric smooth function according to pNi; and e the error. The k-value (basis dimension) of GAMs was set to the minimum kvalue that fitted the curve and explained the data points without over-or under-fitting.

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Most trace metals in seawater are partially bound by organic ligands and their bioavailable "free" concentrations are lower than the total dissolved ion concentrations (Van Den Berg and Nimmo, 1987 be displayed as separate x-axes on the same plot (Fig. 2, 3 and 4). In the Southern Ocean seawater media, the 220 differences between pNi and pNi 2+ are very small due to the assumed low concentration of ligands. In Aquil, however, these differences are large due to the presence of EDTA.  Every strain was able to grow in all Ni concentrations in Aquil media for at least 3 batch cycles. In general, Ni had limited impact on phytoplankton growth rates in the tested range. Six out of the 11 strains displayed statistically significant growth rate changes in response to Ni sensitivity (Fig. 2, Table 2). These strains were Synechococcus  We were interested if we could trust singular datapoints at the extreme ends of the optimum curves, as they often drove trends in our data (e.g., Synechococcus in Fig. 2 at pNi <7.5, total dissolved Ni < 30 nmol/L). Therefore, we did an additional experiment with Synechococcus sp. (CS-205) where we replicated the lowest added Ni treatment (0 nmol/L; 0.07 nmol/L including background Ni) and the optimum Ni concentration (20 nmol/L) ( Table 3). The

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results confirmed the trend in the optimum curve, with the added 20 nmol/L Ni resulting in significantly enhanced growth rates (Table 3).  260 treatment (shown individually). Ni con. is the total dissolved Ni concentration in the media. μ means growth rate (day -1 ). SD means standard deviation. P-value was calculated using T-test. The unit of σPSII is nm 2 reaction centre (RC) -1 .
Ni con.

Photosynthesis performance of phytoplankton
The FRR fluorescence data were largely consistent with the growth rate data in that no strong trends within the Ni 265 range tested were observed for most of species. The σPSII and Fv/Fm measurements across the Ni gradient revealed minimal trends, with generally little variation between treatments ( Fig. 3 and 4). A few exceptions to this general pattern results are mentioned below.    these observations underscore the importance of organic ligands when studying Ni sensitivity of phytoplankton.

Species-specific Ni sensitivity linked to enzyme requirements
Our results are consistent with earlier studies showing that different phytoplankton species have different Nisensitivities (e.g., Glass and Dupont, 2017). Species-specific sensitivities can be due to the different role of Ni as a co-factor for the enzyme SOD, which catalyses the conversion of O2to O2 and H2O2. There are different kinds 390 of SODs, with differing trace metal co-factor requirements. Typically, cyanobacteria utilize either Ni-SOD alone or combinations of manganese (Mn)-and Ni-SOD or iron (Fe-) and Mn-SOD. Diatoms and rhodophytes retain an active Mn-SOD, whereas chlorophytes, haptophytes, and embryophytes have either Fe-SOD or multiple metals associated with minerals considered for OAE should take regional differences of organic ligand concentrations into consideration.

Conclusion
The Ni sensitivity of phytoplankton varied between the 11 species tested within this study but was generally rather low. This may be partly due to the use of nitrate as a nitrogen source in our experiments as other studies have 435 revealed higher Ni sensitivities when growth is fuelled by other nitrogen-sources, such as urea. The reduced sensitivity observed in our study may also be due to the use of the high concentration of organic ligand (EDTA) added to our media, which complexed Ni making it less available for biological interactions. Considering the nitrogen sources, ligand concentration, and phytoplankton composition in test regions is important in assessing the potential environment risks of OAE. Applications of OAE with Ni-rich minerals may be safer in regions with high 440 organic ligand concentrations and low urea concentrations, as this may reduce the impact of Ni on phytoplankton communities.
Appendix A