1194 Chonghong Zhang et al.


Figure 10. a,b: Scanning electron microscopy (SEM) images of early samples; distributed amorphous calcium carbo- nate (ACC) particles and organics in a on 5th day; aggregation of ACC particles and organics in b on 10th day.


diameter of 2−10 μm, an intermediate region of Mg-calcite, and a coat of film composed by ACC phases, calcified bac- teria, and their secreta. To further probe the origin of these spherulites, we collected early stage precipitates and observed them using SEM. Figure 10 shows that crystal nucleation beginswith the formation ofACC, as indicated by corresponding SAED (Fig. 3). In this stage, bacterial cells and biomolecules efficiently bind metal cations (Ca2+ and Mg2+) in solution, triggering carbonate nucleation. More ACC particles are then continuously deposited onto the organic matrix, which grow to 0.2−3 μm in diameter over time. Subsequently, ACC particles gather together by organic adherence to form a sphere-like core with a diameter around 6 μm(Fig. 10b). Interestingly, the size of the core is similar to the size of the central area of mature spherulites observed in the etching experiments mentioned above, and the mor- phology of ACC particles was also comparable. If not coin- cidental, the aggregation of numerous ACC particles, aided by bacteria and biomolecules, evolves into a nucleus of mature spherulite as the mineralization progresses, and remains as the ACC phase even after the formation of stable Mg-calcite spherulites (Fig. 8). Previous studies demon- strated that Mg2+ ions are able to transiently stabilize the metastable phase for relatively long periods (Addadi et al., 2003). In our experiments, Mg2+ ions were a component of the amorphous nucleus (supported by EDS analysis in Fig. 8b), and they slowed the growth of calcite crystallites (Cantaert et al., 2013) such that the Mg-ACC core was stable. Besides, ACC combined with bacterial cells or biomolecules secreted by bacteria over time, and previous studies suggest that organic molecules might be one of the factors stabilizing the ACC phase in solution, which further increases the life- time of the ACC phase in saturated solution. A recent study revealed several important functions of the metastable phase in biominerals and the biomineralization process (Addadi et al., 2003). Concomitantly, the coexistence of the ACC phase and the crystalline phase has been observed in various biominerals (Aizenberg et al., 2003). Limited information is available about the properties of biogenic ACC phases, besides the fact that they are isotropic, and the isotropic properties of the ACC core that has no preferential orienta- tion permits the Mg-calcite subunits to grow outward in all


Figure 11. Scheme of the formation process of Mg-calcite spher- ulite. a: Strain HJ-1 and extracellular polymeric substance (EPS) secreted by bacteria. b: Bacterial cells and EPS trap and concentrate the Ca2+ and Mg2+ ions (+). c,d: Colloidal amorphous calcium carbonate (ACC) particles form with the increasing CO3


2− (−).


e: More ACC particles are deposited onto the plane, and coexistence with bacteria and EPS. f: ACC particles aggregate into sphere-like


core. g: Ambient Mg-calcite subunits grow in a radial mode, and film constituted by calcified bacteria, EPS and ACC particles forms on the surface ofMg-calcite spherulite.


directions (Zhong & Chu, 2010). In this context, the ACC/ organic matter core is crucial to the growth of Mg-calcite spherulites. Although we do not know the specific mechan- ism controlling the growth of Mg-calcite subunits, the sub- units may form via ACC precursors, then grow quickly on account of the higher pH value and supersaturation in the culture medium. Furthermore, the incorporation of Mg2+ into calcite crystals results in lattice strain and selective poisoning of crystal growth perpendicular to the calcite C-axis (Chen et al., 2006), which causes the formation of long Mg-calcite subunits. Thus, we propose a growth mode for Mg-calcite spherulites, as illustrated in Figure 11. Carbonate nucleation begins with the production of the colloidal ACC phase as a result of local supersaturation. The two-dimensional solid plane gradually forms as more col- loidal ACC particles are deposited, due to strain induced by bacteria or bacterially excreted biomolecules. Subsequently, more ACC particles are deposited onto the plane, and aggregate to form a more stable sphere-like core. This core subsequently acts as a nucleus for assembling the Mg-calcite


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