**1. Introduction**

Calcium carbonate (CaCO3) is the most important biogenic mineral, in terms of quantity, global distribution, and diversity [1]. The production of CaCO3 provides a number of ecological goods and services, such as shoreline protection and habitat structures. For example, coral reefs are one of the most important living bioconstructions of CaCO3 [2] harboring one-quarter to one-third of all marine species [3], and thus serving to be socially and economically important [4]. Unfortunately, future projections show marine biomineralization will become severely impacted by ocean acidification (OA) due to the reduction of carbonate ion concentrations in the oceans [5,6].

Corals calcify extracellularly in a fluid that is separated from the seawater by at least two cell layers [7,8] and rely on a number of active and passive ionic exchanges. For example, calcium ions are actively transported into the extracellular calcifying fluid (ECF) by the epithelium cells of the coral polyp [9,10] while protons are removed [11], establishing favorable conditions for the precipitation of CaCO3 [12]. Similarly, carbon either diffuses into the ECF as carbon dioxide [CO2] or is actively transported into the ECF in the form of bicarbonate [13,14]. Some coral species can calcify in ocean water that is undersaturated with respect to aragonite [15], whereas other species cease to grow and vanish [16,17], which demonstrates a range of biological controls governing the mineralization process. Therefore, to understand which marine calcifiers will be affected by future reduction in ocean saturation states and to estimate its implications for the global carbon cycle, we need to explore a range of possible ECF scenarios.

**Citation:** Reymond, C.E.; Hohn, S. An Experimental Approach to Assessing the Roles of Magnesium, Calcium, and Carbonate Ratios in Marine Carbonates. *Oceans* **2021**, *2*, 193–214. https://doi.org/10.3390/ oceans2010012

Academic Editor: Peter Schupp

Received: 30 August 2020 Accepted: 22 February 2021 Published: 3 March 2021

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The significance of biologically-induced and biologically-influenced mineralization is irrefutable. For example, the skeletal organic matrix [SOM] within corals is considered a major factor controlling the precipitation of CaCO3. A number of studies have reported that the SOM contains not only acid-rich proteins (e.g., sulphated proteoglycans), but also assemblages of adhesion and structural proteins, which together are thought to provide a template for aragonite precipitation [10,18–20]. Additionally, the dissolution and precipitation of CaCO3 in aqueous solution is largely dependent on abiotic factors relating to the saturation state (Ω) of the ECF [21,22], which is defined by the product of the dissolved ions forming the mineral divided by the stoichiometric solubility product, Ksp\* (Equation (1)).

$$\Omega = [\text{Ca}^{2+}]^\* [\text{CO}\_3^{2-}] / \text{K}\_{\text{sp}}\text{ }^\circ\text{.}\tag{1}$$

As expressed above, Ω is an extremely useful indicator of the equilibrium or disequilibrium of a solution with a mineral surface. When the ion product, [Ca2+]\*[CO3 <sup>2</sup>−], equals the solubility product, Ksp\* , the saturation state equals one and the system is in equilibrium. If the saturation state is below one because the ion product is lower than the solubility product, the solution is undersaturated and the mineral dissolves. A saturation state higher than one indicates supersaturation where the product of the ion concentrations is greater than the solubility product. In this case, it is thermodynamically viable that dissolved ions precipitate into a crystal structure [22]. The observation that the precipitation rate of CaCO3 increases with an increasing saturation state [21,23–27] has led to the development of empirical relationships (Equation (2)) that describe the calcification rate, G, as a function of the saturation state, where *k* is the reaction rate constant and n is the empirical reaction order.

$$\mathbf{G} = k^\* [\Omega - 1]^\mathbf{n},\tag{2}$$

This equation has been applied to predict calcification rates in corals [28] and has successfully been used to simulate the dynamics of ion concentrations in the calcifying fluid of corals and coccolithophores [14,28–30]. However, Equation (2) appears to ignore the theoretical basis shown in Figure 1, which emphasizes how the product of varying calcium and carbonate ion concentrations can obtain the same Ω value. This is also supported by [31], which demonstrated how the rate of calcite precipitation differed due to the ratio of calcium to carbonate despite having the same oversaturated Ω. Although Ω is a good predictor of dissolution and precipitation of CaCO3, it does exclude the possibility that ion concentrations differ while obtaining the same Ω and could therefore account for observational variations among marine calcifiers.

**Figure 1.** Varying concentrations of calcium and carbonate ions [Ca2+:CO3 <sup>2</sup>−] at fixed Ωara of 25, 15, and 5. This demonstrates the underlining principle that calcium and carbonate ion concentrations can obtain the same Ωara value at different Ca:CO3 ratios, therefore questioning the empirical equation that prescribes the calcification rate as a function of Ωara alone.

Based on this rational, we decided to incubate coral skeleton fragments under six controlled abiotic chemo-static scenarios. By emulating previously measured ECF conditions, we kept all the solutions oversaturated in respect to Ωara = 10, pH 8.7, and maintained a typical tropical temperature of 25 ◦C. Experiment 1a recreated a magnesium [Mg] free condition (strong Mg removal activity) with a high Ca:CO3 ratio (e.g., no dissolved inorganic carbon (DIC) concentrating mechanism and a weak proton removal from the ECF), while experiment 1b recreated a Mg-free solution with a low Ca:CO3 ratio (e.g., mimicking a DIC concentrating mechanism resulting in DIC concentrations three times greater than ambient seawater and a strong proton removal from the ECF resulting in elevated total alkalinity (TA) four times greater than ambient seawater). Experiment 2a recreated a medium Mg scenario (representing concentrations half that of the modern seawater) with a high Ca:CO3 ratio, while experiment 2b recreated a medium Mg scenario with a low Ca:CO3 ratio. Experiment 3a recreated a high Mg scenario (equal to that of modern seawater, i.e., no active removal of ions from the ECF) with a high Ca:CO3 ratio, while experiment 3b recreated a high Mg scenario with a low Ca:CO3 ratio.
