The biogenic mineralization of calcium carbonate (CaCO3) by H. halophila in the presence of zinc was investigated. H. halophila maintained the ability of CaCO3 mineralization in a Zn2+ contaminated environment. The bacteria function as inducers for the mineralization of calcium carbonate mineralization since in media without bacteria no mineralization of CaCO3 was observed. The inorganic phase was mineralized in media with high calcium concentrations only. The basal Ca2+ concentration (around 4 mg/L) in the MH medium, which has no calcium supplementation, is not sufficient to mineralize calcium carbonate in high amounts. Bacteria that are cultured in MH medium show the formation of isolated particles on the cell surface. Spontaneous mineralization of calcium carbonate in medium in the absence of H. halophila was not observed in the experimental setup. H. halophila which was adapted to zinc in the medium still mineralizes calcium carbonate. In the presence of Zn2+ the formation of calcite was suppressed, which is mineralized in MH 2 and MH 4 media, instead vaterite and monohydrocalcite were synthesized (Table 2). Functional groups on the cell surface may lead to changes in nucleation energy and thus induce the mineralization process . The onset of significant mineralization activity that is associated with the decrease of calcium from the medium was determined for cultivation times longer than 3 days. Within the first 6 days the major decrease of calcium from the medium was monitored. In parallel, the accumulation of membrane associated calcium increases significantly from day 7 onwards which is indicative for the mineralization of CaCO3. A similar time frame for the mineralization of calcium carbonate by Flavobacterium was reported . Flavobacterium strains show the induction of calcium carbonate mineralization between 3 and 7 days of cultivation in medium with 7.5% salt concentration at 22°C. The mineralization process of Flavobacterium and H. halophila both result in the formation of calcite .
Beside the mineralizing organism, the mineralization process is highly dependent on environmental conditions, e.g. temperature, pH, ionic strength . Various microorganisms show different optimal mineralization conditions. High salt concentrations negatively affect the biomineralization process of H. halophila and other moderately halophilic bacteria strains (e.g. Flavobacteria, Acinetobacter) . Interestingly, other moderately halophilic bacteria strains have an optimal external salt concentration of 10% to 20% for the biomineralization of calcium carbonate and the formation of inorganic crystals is suppressed under low salt concentrations . This indicates that beside environmental conditions bacteria actively influence the mineralization process. Furthermore, Hammes and Vestraete  stated that microorganisms can influence most mineralization factors, e.g. pH, local calcium concentration by surface adsorption, concentration of dissolved inorganic carbon, and therefore have some control over the biomineralization process. One of the influencing parameters is the pH value in solution. The pH value in the media increased to alkalinity (around pH 9) during the cultivation of H. halophila. The bacterial metabolic process generates a global alkaline environment. In particular the metabolism of organic nitrogen, like the aerobic oxidative deamination of amino acids and the reduction of nitrate, leads to the increase of the pH value in the surrounding environment . The pH shift of the medium from neutral to alkaline conditions facilitates the precipitation of calcium carbonate. Moreover, bacteria cells have been reported to act as nucleation sites or sites for calcium accumulation . Positively charged ions (e.g. Ca2+) can be accumulated at negatively charged functional groups on the cell surface and subsequently react with anions to form insoluble materials like calcium carbonate . The experimental set up showed that the amount of surface bound calcium depends on the initial Ca-acetate concentration in the medium. High calcium concentrations in the medium leads to a high membrane associated calcium concentration. Zn2+ in the medium does not influence the accumulation of Ca2+ on the bacteria cells which is in accordance to the continuity of the biomineralization process in the presence of zinc.
The soluble intracellular calcium concentrations were similar in medium with low and high Ca2+ concentrations. In contrast to the membrane-associated calcium concentrations, zinc apparently influences the intracellular calcium concentration. In both media containing Zn2+ (MH 2Z and MH 4Z) the concentrations of calcium was significantly increased in the cell lysate compared to the corresponding media without Zn2+. The levels of cytoplasmic free Ca2+ are strictly regulated in bacteria cells, since it is assumed to play a role in chemotaxis, cell division and signal transduction . Ca2+ levels in E. coli cells range between 200 to 300 nM with 2 to 7 fold fluctuations on external calcium concentration changes . Interestingly, bacteria cells in the stationary phase appear to have less control over internal free Ca2+. The cytoplasmic Ca2+ levels can be regulated with transporter systems like the pH dependent Ca2+/H+ antiporter  or the inorganic phosphate co-transporter . Also, a polyhydroxybutyrate-polyphosphate (PHB) complex in E. coli was reported that can accumulate large amounts of Ca2+ in addition to function as a specific Ca2+ channel . Our results suggest that zinc ions affect Ca2+ homeostasis leading to high intracellular calcium concentrations (Figure 5). The effect might be based on interference of Zn2+ with calcium transporter systems which regulates the intracellular calcium levels.
Zinc was only detected in the medium or on the surface of bacteria cells. The levels of zinc in lysed bacteria cell samples were below the detection limit. The main fraction of the zinc ions in solutions was accumulated on the bacterial cell surface by biosorption and removed from the environment leading to the depletion of zinc in the medium. Contrasting this extracellular immobilization of zinc ions, microalgae incorporate zinc and decontaminate it by the formation of zinc-phosphate based nano needles . Although zinc is required for many processes in living organisms high intracellular zinc concentrations are toxic. Therefore, various cellular systems have evolved to maintain zinc homeostasis in bacteria cells. In bacteria members of the HME-RND (heavy metal efflux - resistance, nodulation, cell division) protein family, CDF (cation diffusion facilitators) family, and P-type ATPases were identified which are involved the export of Zn2+. Furthermore, the efficient cell surface binding of zinc ions might also contribute to low intracellular Zn2+ levels. Based on the bacterial sorption model of Fein et al. acidic (pKa < 4.7, e.g. carboxyl and phosphodiester), neutral (pKa ≈ 7, e.g. phosphomonoester) and basic sites (pKa > 8, e.g. hydroxyl and amine groups) are involved in metal binding . The Zn2+ adsorption was reported primarily to carboxyl- and phosphate-type functional groups . Zinc homeostasis mechanisms and cell surface binding of zinc may be responsible for maintaining intracellular zinc concentrations below the detection limit.
Calcite was the predominant polymorph which was mineralized in media in the absence of Zn2+. The formation of calcite in media containing NaCl as sole salt is in agreement with earlier reports [27, 28]. The mineralized polymorph is not only dependent on environmental conditions (e.g. ionic strength, pH, temperature) but is also dependent on the biomineralizing bacteria strains . Under the same experimental conditions Flavobacterium and Acinetobacter stains mineralized other CaCO3 polymorphs. Furthermore, the Ca-acetate concentration in the media showed no effect on the mineralized polymorph, calcite was predominantly mineralized. Surprisingly, in MH 2 medium monohydrocalcite, which is thermodynamically less stable than calcite, was detected at 22 days of cultivation. In precipitation experiments without organic material, monohydrocalcite precipitates at a solution saturation state which is significantly lower than the saturation state of solutions precipitating calcite . Furthermore, the precipitation of calcite promoted the dissolution of monohydrocalcite, suggesting the transition of monohydrocalcite to calcite . In summary, the mineralization of monohydrocalcite starts at low Ca2+ concentrations and precedes the formation of calcite which is mineralized after the accumulation of high amounts of Ca2+. Our biomineralization experiments did not show in general a periodic change of mineralization products. Rather monohydrocalcite is stabilized either by zinc ions or by organic–inorganic interfacial interactions. In medium with Zn2+ the mineralization of calcite was suppressed. The predominant polymorphs were vaterite and monohydrocalcite. Under non-biological conditions, vaterite transforms quickly into calcite, which is the more stable phase of calcium carbonate. Using the double diffusion technique for the synthesis of calcium carbonate in the absence of organic additives at pH values between 10.4 to 10.8, calcite was generated while in the presence of Zn2+ aragonite was precipitated . The Zn2+ cations were assumed to inhibit the transformation of the aragonite to the stable polymorph calcite [30, 31]. The biomineralization in the absence of zinc resulted in the mineralization of calcite, similar to the synthesis in the absence of bacteria, while in the presence of zinc monohydrocalcite and vaterite were generated. Since in double diffusion experiments aragonite and not monohydrocalcite or vaterite was precipitated, our results indicate that the bacteria additionally influence the mineralized polymorph. It was also reported, that natural deposits of vaterite are most often associated with biogenic activity . Organic molecules might stabilize and/or favor the vaterite formation due to (1) organic templates that induce the heterogeneous vaterite mineralization  or (2) the action of organic molecules that inhibit the transformation of the metastable vaterite to stable phases . The mineralization and stabilization of the less stable CaCO3 polymorphs in our experiments might be accounted on these phenomena, too. In Bacillus licheniformis S-86 cultures, the extracellular polymeric substance (EPS) induces the agglomeration of bacteria cells in solution , which was also monitored in the cultivation of H. halophila in our experiments. In mineralization experiments with B. licheniformis S-86 producing EPS as well as in EPS solutions without bacteria calcite was mineralized. It was proposed that dissolved organic carbon (DOC) released from the EPS complexes Ca2+ ions in solution which reduces high supersaturation states which favor the formation of vaterite to lower Ca2+ levels which enhance the precipitation of calcite .
The morphology of the mineralized inorganic particles is divers, exhibiting globular, sponge-like, and triangular shapes. Interestingly, no defined inorganic structure can be correlated to a distinct CaCO3 polymorph. This was also shown for biogenic mineralized calcite and aragonite polymorphs which were morphologically not discriminable by electron microscopy . The generated calcite agglomerates in MH 2 medium, consisting of triangular platelets exhibit a smooth inner part (Figure 7B). This region might be attributed to localization sites of bacteria, which initiated the mineralization process and became embedded during the mineralization process. Similar defects in biomineralized calcium carbonate crystals were reported . Calcium carbonate crystals were pitted by bacteria-shaped holes which were assumed to be formed as a consequence of the deposition of mineralization products on the cell surface.
The biomineralization of calcium carbonate and also other inorganic materials can be classified into two different processes: (I) biologically induced and (II) biologically controlled mineralization . The two processes differ regarding the degree of biological and genetic control. The mechanism (II) is generally more strictly regulated. The microalgae Scenedesmus obliquus mediate extracellular calcite formation in a biologically induced mineralization process. In the presence of Zn2+ the calcite polymorph is suppressed and aragonite is synthesized . Zinc ions affect the biomineralized CaCO3 polymorph in both, algae and halophilic bacteria. Organisms mineralizing CaCO3 under biological control are e.g. gastropods and sea urchin larvae. There the mineralization takes place in a confined compartment inside the cell and organism, respectively. The mineralization of nacre in gastropods, a highly structured assembly of aragonite platelets and organic components, is controlled by organic template structures and soluble proteins [reviewed in . The template forms a compartment of equally spaced layers in which the aragonite is mineralized. The organic molecules strictly regulate the polymorph, morphology, and nucleation of the inorganic material. In sea urchin larvae, spicules (skeleton) are synthesized in primary mesenchyme cells (PMC) starting from the 16-cell stage . In early stages the skeleton consists of amorphous calcium carbonate which is stabilized by proteins. In the further development the amorphous phase transforms into calcite . In the presence of Zn2+ the spicule formation is suppressed . Compared to the biologically induced mineralization process in halophilic bacteria and algae, zinc has a fatal effect in spicule formation. For cadmium, gold, and silver it was suggested, that biominerals play also a role in detoxification processes by immobilization of adverse ions . Here, we showed that for zinc contaminations the biologically induced mineralization in halophilic bacteria have a similar effect.
The mineralization of inorganic materials by moderately halophilic bacteria can be specifically modulated in the presence of zinc ions. These investigations show that bacterial mineralization processes might be exploiting for applications, like the remediation of wastewater or the generation of functional materials for technical use.