Synthesis Experiments

In vitro synthesis experiments were conducted in polyethylene bottles at room temperature (20 °C). Eight different amino acids were tested to probe the effect of different amino acid properties, such as polarity, hydrophilicity, and acidity, on carbonate formation (Table 1). Amino acids (7.5 mM) and chitin (0.025 g/L) were mixed into 50 mL of deionized (DI) water and continuously stirred in polyethylene bottles for half an hour. Then, 6.25 mM calcium chloride dihydrate (CaCl₂·2H₂O), 31.25 mM magnesium chloride hexahydrate (MgCl₂·6H₂O), and 25 mM sodium bicarbonate (NaHCO₃) were dissolved into the organic substrate bearing solutions. After another 30 min of mixing, to ensure that everything had dissolved, the bottles were sealed and kept at room temperature. After a week, solutions were filtered and air-dried.

In a separate set of experiments to test the effects of solid surfaces on Ca–Mg carbonate precipitation, aspartic acid, glutamic acid, and chitin were first individually mixed with deionized (DI) water. Natural amorphous opal-A (opal) slices from Australia, glass slides (GS), and gold-coated glass slides (GCGS) were then placed into these solutions for 24 h with closed lids to allow solutes to adsorb onto their surfaces. Next, calcium chloride dihydrate (CaCl₂·2H₂O) and magnesium chloride hexahydrate (MgCl₂·6H₂O) were added to the solutions to provide the Ca and Mg needed to precipitate Ca–Mg carbonate minerals (see Table 2 for solution concentrations). In most solutions, the initial Mg/Ca ratio was set to 5 as an analogue to the seawater Mg/Ca ratio (∼5); however, some solutions had lower Mg/Ca ratios of 3–2 (Table S1). Absolute concentrations of Mg range from 40 to 20 mM (Table S1). Polyethylene bottles containing these solutions (100 mL each) were placed into a sealed desiccator with ammonium bicarbonate ([NH₄]HCO₃) powders (20 g), allowing for NH₃ and the necessary CO₂ to diffuse into the open-lid solutions for the carbonate minerals to precipitate. After one week, the solutions were removed from the desiccators, and precipitates were filtered out with 20–25 μm pore-size filter papers. Surface materials, i.e., opal, GS, and GCGS, were picked out and rinsed with DI water for 30 seconds to remove precipitates that had been formed in the solution but settled on the surfaces, leaving behind only the minerals that precipitated directly onto the solid surfaces.