Nucleation and Growth of Mg-Calcite Spherulites 1191
double-distilled water and dropped onto a copper mesh. The dried sample was stained with phosphotungstic acid for 30 s, and dried before being submitted to TEMimaging. Precipitates collected by centrifugation were analyzed using an EVO 18 scanning electron microscopy (SEM) instrument (ZEISS, Jena, Germany) equipped with an energy dispersive spectro- scope (EDS) for imaging minerals. Dried samples were coated with 8nm of gold before undergoing SEM imaging. Samples were further investigated by TEM and selected area electron diffraction (SAED) using a Tecnai electron microscope (FEI Co., Hillsboro, OR, USA). Phase characterization of the pre- cipitates was determined using a D/max-B (III) X-ray diffrac- tion (XRD) with Cu-Kα radiation. The scan rate of 0.01°/s was applied to record the patterns in the 2θ range between 10° and 60° at 35kV and 20mA. Qualitative identification of mineral phases was performed using the Jade 5.0 program. IR spectra were measured on a Bruker Vector-22 Fourier transform infrared (FT-IR) spectrometer at room temperature.
RESULTS AND DISCUSSION
Strain HJ-1 grew well during the 50 day biotic experiments, and the cell density increased rapidly in the initial stages and reached a maximum on day 10 (6.0×109 cfu/mL), then decreased slowly thereafter (Fig. 2). In the meantime, the turbidity of the solution increased noticeably upon con- tinued incubation, indicating fewer particles present in the
suspension as time proceeded. Precipitation then occurred gradually, and large quantities of solid material adhered to the bottle wall. By contrast, no precipitate was formed in the abiotic (control) experiments. Thus, strain HJ-1 was essential for inducing calcite precipitation in this system. Strain HJ-1 appeared to alter the physicochemical parameters of the system to promote carbonate precipitation. In contrast with abiotic experiments, in biotic experiments, bacterial respiration and degradation of organic matter by bacteria result in the release of CO2 and NH3, and released ammonia increased the pH, which increased carbonate ions in the culture medium, as indicated by the following equilibria: CO2(g)+H2O(l)↔H2CO3(aq)↔HCO3(aq)
+2H(aq) + . As expected, carbonate precipitation occurred
− +H(aq) + ↔CO3(aq)
in the presence of Ca2+ ions as soon as the culture medium became supersaturated. Furthermore, the bacteria cell wall contains a number of negatively charged surface functional groups, such as hydroxyls, carboxyls, and phos- phates, and bacterial EPS, which includes carboxylic and amino functional groups, are also known to have a high Ca2+/Mg2+-binding capacity, and to serve as nucleation templates for carbonate particles (Rodriguez-Navarro et al., 2007). Analysis of XRD patterns confirmed that the precipitates
present between days 30 and 50 were pure Mg-calcite. Since XRD patterns of precipitates obtained during the early stages of the experiment displayed no obvious diffraction peaks, TEM analysis was also carried out. Figure 3 shows a representative TEM image of these precipitates and the corresponding SAED pattern of early aggregates, which could be indexed as amorphous calcium carbonate (ACC) phases, conforming their status as mature Mg-calcite particles derived from ACC. Analysis of Mg-calcite samples from day 30 to day 50 by SEM revealed the coexistence of hemi-spherulites and spherulites, which accounted for more than 90% of precipitates with differ- ent morphologies (Fig. 4). Fragments of Mg-calcite spherulites can be used to investigate the interior structure of these crystals, and the results implied that the spherulites may be composed of a nucleus surrounded by numerous needle-like subunits. As shown in Figure 4, the outer surface of spherulites
Figure 2. Growth curve of strain HJ-1 in M2 liquid media.
and the bottom surface of hemi-spheruliteswere consistently covered by a film layer. Interestingly, we serendipitously
2−
Figure 3. Transmission electron microscopy image (a) and corresponding selected area electron diffraction pattern (b) of solid precipitates on day 10. The diffuse rings correspond to amorphous calcium carbonate.
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