1190 Chonghong Zhang et al.


remains poorly understood. In particular, the origin and phase composition have received minimal attention. Monocrystals and polycrystals tend eventually to form spherulites in microbial systems, since organic acids and polysaccharides promote spherulite formation (Mercedes- Martín et al., 2016). Mechanisms of spherulite aggregation have also received considerable attention. Ocaña et al. (1995) concluded that aggregation can occur in either an undirec- tional or omnidirectional manner, with unidirectional aggregation resulting in the formation of spherulites. Gránásy et al. (2005) claim that spherulitic particles form by growth front nucleation when new crystal grains nucleate at surfaces with a different lattice orientation to the parent crystal, through a randomized orientation, which accounts for the isotropy of spherulitic growth at large length scales and long times. The evolution of spherulitic particles has been proposed to take place in two different ways: (a) Spherulites arise from a central precursor via multi- directional growth or (b) they form from a precursor in which the edges fan out, giving rise to dumbbell-like shapes which might ultimately result in a spherical shape (Gránásy et al., 2005). Morphological control of crystals is important in industrial processes (Wang et al., 2005). If certain bacterial strains or metabolic states display distinct spherulite- forming characteristics, unique morphologies, or mixtures of morphologies may be selected to prepare materials for technical applications. Previous biomineralization studies on CaCO3 demon-


strated that Mg2+ ions contribute to the formation of calcite spherulites by crystallization from solution (Cantaert et al., 2013; Xu et al., 2017). This could be due to slow crystal- lization in highly viscous environments containing Mg2+ ions. Herein, experiments involving Curvibacter lanceolatus strain HJ-1 and calcium/magnesium saltswithoutCO3


2− were


performed over 50 days, which resulted in interesting radial Mg-calcite spherulites. We investigated their interior struc- ture and mineral composition using etching experiments, and time-resolved experiments provided further insight into their formation. Our aim was to elucidate the nucleation patterns and crystallization processes involved, and deter- mine the spherulitic composition and structure. In addition, we hoped to identify factors controlling calcite morphology, and discover how crystallization parameters influence par- ticle morphology in bacterial systems containing Mg2+ ions.


EXPERIMENTAL SECTION


Bacterial Identification and Characteristics Curvibacter lanceolatus strain HJ-1 was obtained from alfisol samples from a test field (N32°01′, E118°51′) in Nanjing Agricultural University. Strain HJ-1 was chosen for experi- ments in our study because (1) it grew quickly and produced large amounts of EPS when grown in M2 medium and (2) it is a soil microbe that was shown to precipitate spherulitic calcite particles in our preliminary experiments. Strain HJ-1 was defined as Curvibacter lanceolatus (AB681931) after


analysis of the 16S rRNA gene sequence (Li et al., 2017). Strain HJ-1 colonies on solid media were white, transparent, or rose-colored, with a smooth surface, and a diameter of ~1mm. Gram staining confirmed strain HJ-1 as Gram- negative. Transmission electron microscopy (TEM) images of bacterial cells revealed a rod-like cellular morphology with approximate dimensions of 0.5×2 μm (Fig. 1).


Crystallization Experiments


Strain HJ-1 was used to inoculate a sterile liquid culture containing tryptone (10 g), beef extract (2 g), and NaCl (5 g) in 1 L of deionized water and incubated for 24 h (to late exponential phase) at 30°C (the optimal growth temperature for strain HJ-1) in a shaker with shaking at 180 rpm. Bac- terial calcite mineralization experiments were carried out in sealed Erlenmeyer flasks (150 mL) containing inoculation culture (10mL) and M2 medium (90 mL) at 30°C under quiescent conditions in an incubator. The M2 medium for carbonate precipitation was composed of 1% (mass fraction) proteose peptone, 0.05MAnalar-grade calcium acetate, and 0.15M Analar-grade magnesium acetate. The initial pH of the solution was adjusted to ~8 by addition of 1M NaOH and measurement using a pH meter. We observed con- tinuous precipitation in M2 medium, and strain HJ-1 grew well under these conditions. Solid samples in flasks were collected by centrifuging for 10 min at 5,000 rpm. After air drying, minerals were prepared for mineralogical and mor- phological analysis. All experiments were carried out in tri- plicate.We also performed abiotic control experiments.


Observations and Measurements


Bacterial density in the medium was measured using plate count method. An H-7650 TEMinstrument (Hitachi Corp., Tokyo, Japan) was used to observe the bacterial cellular morphology. Several bacterial colonies were suspended in


Figure 1. Transmission electron microscope image of a cell of Curvibacter lanceolatus strain HJ-1 grown on the surface of solid agar medium.


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