*4.3. Polynucleation and Spontaneous Nucleation*

The ratio of calcium to carbonate clearly matters within the ECF, as it has been shown to describe the rate and morphology of CaCO3 (Figures 3 and 4). Precipitation pathways can be either direct or sequential depending on the free energy available on the surface as determined by pre-nucleation clusters (PNC), growth, and transformation [64–67]. Polynucleation occurred in all scenarios in this study, which led to a complex situation increasing the number of active sites on the surface layer, and therefore a stronger dependence on supersaturation than solely the layer-by-layer mineralization process [31]. Further complications arise because the rate-determining step may change with time as the number of defects and the relative dimensions of the crystal faces become modified during precipitation. Therefore, there are often deviations from the idealized kinetic models, as there may be a number of mechanisms operating in concert [66–68]. It is interesting to note

that spontaneous nucleation appeared in the experiment with high excess of calcium ions relative to carbonate ions (i.e., the ambient DIC and TA seawater treatment) but not in the low Ca:CO3 treatment (i.e., the DIC concentrating mechanism scenario), however previous studies have shown that PNC usually form in a low Ca:CO3 solution, equivalent to the binding of ions during crystal formation [47]. This may be due to metastable conditions under which precipitation of the mineral is delayed despite the solution being oversaturated in respect to Ωara [67].

The higher calcification rates in the Mg-free and 47:1 Ca:CO3 scenario were obtained primarily due to spontaneous nucleation within the incubation chamber leading to much higher precipitation and thus greater reactive surface area. Previous calcite precipitation experiments in supersaturated (Ωcalcite 5, 16; pH = 10; T = 20 ◦C) conditions did not produce spontaneous nucleation and showed an optimum precipitation rate when Ca:CO3 = 1:1 [31]. However, there are differences between this study and [31], one of which is the use of NaHCO3 to prepare the carbonate solution in this study instead of K2CO3. This together with a temperature difference of 5 ◦C can potentially explain the variation between our observations and [31].
