The BCC produced in presence of B. subtilis had different morphological and crystallographic properties, depending of media composition: a) conditions where the calcium source is not associated to carbon one, non-coupled (GYL and GLC); or b) where the calcium is attached to the carbon source, coupled (LAC and ACE). The predominant morphologies in petri dish were mineralized colonies with rounded shape and convex (Fig. 1), in contrast to experiments done in liquid media were dumbbell, rhombohedral and spherulitic shapes are common (Han et al. 2019). Independently to media, the main factor that induced carbonate precipitation is the microenvironment alkalinization, that enhance complexation of Ca+ 2 free ions with free CO3 − 2 ion (Dupraz et al. 2009). Specifically, in nutrient media, glutamine deamination (Dervaux et al. 2014), and carbonic anhydrase activity (Frankel and Bazylinski 2003; Perito and Mastromei 2011; Oppenheimer-Shaanan et al. 2016; Han et al. 2019) could provoke this pH changes (Jiménez-Delgadillo et al. 2018). pH changes in long term Bacillus culture in agar-rich media (Robinson et al. 1991).
Only BCC-GLC were orange pigmented (Fig. 1b), which is related to iron chelation by pulcherriminic acid produced in biofilms of carbohydrates supplemented media (Arnaouteli et al. 2019).
Thin section micrography show birefringence phenomena under polarized light (Fig. 2). However, due the presence of optical interference color of light green-extinction characteristic 4th order birefringence it can be interrelated has arrangement of typical calcite carbonate crystals (Aizenberg and Hendler 2004). However, did not show the characteristic calcite cleavage. Difference in the interference color in crystals indicates a non-oriented arrangement of microcrystal growth, it can be produced by the randomized growth of bacteria colonies. However, mineral growth in glycerol media is also determined by crystal nucleation in EPS produced by cells (Oppenheimer-Shaanan et al. 2016), that is probably the cause of its amorphous habit. Micrite is common in marine sediments and microbialites (Perri and Spadafora 2011) with spherical and radial-fibers growth as we observed in all BCC; mineralized cells are also present (Rasmussen and Muhling 2019).
Several strains of Bacillus could mineralize different polymorphisms of CaCO3, such calcite and vaterite (Seifan et al. 2016; Andrei et al. 2017; Huynh et al. 2017), but factors that induce only one kind of polymorphism are not jet understood. In this work well only observe calcite production, even if calcium-carbon source was non-coupled. Other authors have identified this crystalline phase in B. subtilis (Zhuang et al. 2018; Han et al. 2019). The obtained BCC had different crystallinity (BCC-GLY < BCC-ACE < BCC-LAC < BCC-GLC), possibly related to mineralization process in each formation-condition (Fig. 3b). The IC of BCC-GLC may be similar to abiotic process pH, Ca2+ concentration and nucleation sites are critical (Perito and Mastromei 2011). For coupled calcium-carbon sources, like ACE (Fig. 4d, S5), the mineralization mainly occurred over cellular structures such as cell wall and EPS (Marvasi et al. 2012; Dhami et al. 2013). In that sources, critical Ca2+ concentration could be accumulated in cell wall because the efflux pump and specific channels (Saier et al. 2002). The membrane and cell wall components could be nucleation sites because its negative charges of teichoic acid that attract Ca2+ (Perito et al. 2014).
Lower IC in BCC-LAC and BCC-GLY could be associated to higher production of organic matter or amorphous calcium carbonate (ACC). This is the polymorphic precursor of crystalline structures under biotic or abiotic mineralization (Bots et al. 2012; Cantaert et al. 2016). Some interaction with glycoproteins or organic molecules increased the ACC stability and prevent spontaneous crystallization (Aizenberg et al. 2002; Weiner et al. 2003). Additionally, in non-coupled calcium-carbon sources (GLY and GLC), BCC nanodeposites with the same sizes of calculate crystallite were evident over calcite scales and mineralized EPS (Fig. 4a, 4b, S4). This is the first work where such kind of precipitants are observed in biocomposites of B. subtilis.
The main components of B. subtilis EPS, that promote calcite aggregation, are TasA and TapA amyloid proteins and exopolysaccharides (Azulay et al. 2019); moreover, there are reports of nanotubes formation when B. subtilis grows in rich media (Bhattacharya et al. 2019). In this study, both structures were observed in BCC-GLC (Fig. 5b) and BCC-LAC (Fig. 5c), respectively.
In order to understand the BCC composition, TGA and DSC analysis were performed (Fig. 6). Thermic change of biotic and abiotic vaterite was reported with an exothermic peak between 317º − 318º C, that was associated to organic matter decomposition with CO2 and NO2 release (Rodriguez-Navarro et al. 2007). Furthermore, at 700º − 800º C a second reaction occurs, from CaCO3 to CaO (Al Omari et al. 2016). As we saw, BCC-GLY presented greater abundance of organic matrix; however, temperature decomposition not follow enthalpy rise. Some organic acids could change temperature peaks of that process (Li et al. 2018).
Mineralization degree (BCC-ACE > BCC-LAC > BCC-GLC > BCC-GLY) also affected thermic stability of BCC in relation to enthalpy and decomposition temperatures (Fig. 6). This may be to mineral abundance or lattice variation (Pokroy et al. 2006), such as in BCC-GLC with higher crystallinity but low presence of CaCO3.
In BCC, EPS could increase complexation by the interaction of the ions with the electronegativity charge of its expose functional groups (Ercole et al. 2007; Oppenheimer-Shaanan et al. 2016). This explain the random nucleation of minerals that occurs in the bacteria microenvironment. However, in BCC-GLY the synthesis level of EPS increases the amount of nucleation sites but affected negatively the crystallinity. EPS have a key role in CaCO3 precipitation (Arias and Fernández 2008), and B. subtilis produce high amount of different types in glycerol supplemented media (Oppenheimer-Shaanan et al. 2016). Besides, EPS is important to CaCO3 precipitation (Decho 2010; López-Moreno et al. 2014), because it attract Ca2+ to its chemical functional groups, such as: COO-, NH3-, PO4- and SO4- groups (Schmitt and Flemming 1998; Humbert and Quilès 2011). Those group, related to main EPS components (exoplysaccharides and proteins) play a key role in BCC formation (Azulay et al. 2018). P content may be related to extracellular DNA usually present in EPS (Peng et al. 2020).
In this study the BCC produced in presence of B. subtilis were constituted manly by calcite. We demonstrated that the quality of this biogenic calcite was only influenced by calcium-carbon source. In that sense, the best composites were obtained when bacteria were grown in supplemented medium by glucose and calcium chloride. Finally, this non-coupled calcium-carbon condition promotes several nucleation sites for CaCO3 precipitation, and it was no need to provide with urea to achieve the necessary alkalinity.