Calcium carbonate (CaCO₃) makes up almost 4% of the Earth’s crust and has been studied extensively due to its importance in biomineralisation in natural environments, including carbon cycling, alkalinity generation, and the biogeochemical cycling of elements^1–3. In addition to its importance in nature, however, the enhanced mechanical properties of certain biotic calcium carbonates have inspired many studies to try to understand their structural secrets^4–6, and prompted their use in geotechnical engineering to improve the mechanical response of soils^7–11. A clear example of these natural materials is nacre in mollusc shells, formed of microlaminate composites of aragonite and/or calcite, each with an associated organic matrix that gives them a fracture toughness 3000 times greater than that of the constituent mineral alone¹². Clearly, the coupling of a mineral phase with an organic material (i.e. biomineral) plays a vital role in the formation and stabilisation of the final CaCO₃ precipitate, in addition to contributing to its ultimate mechanical properties^4,13–17. However, understanding the interrelationship between biotic precipitation, polymorphism, and long-term stabilisation has proven to be far from trivial.

It is well established that CaCO₃ has three polymorphs: vaterite, aragonite, and calcite, with rhombohedral, orthorhombic and hexagonal structures respectively, in order of decreasing solubility and increasing thermodynamic stability¹⁸. Additional metastable forms have been noted in the literature, all of which are hydrated: monohydrocalcite (CaCO₃·H₂O), ikaite (CaCO₃·6H₂O), calcium carbonate hemihydrate (CaCO₃·1/2H₂O), and amorphous calcium carbonate (ACC)^19,20. Thus far, most studies have tackled the formation and crystallisation of CaCO₃ in abiotic systems, with a particular focus on ACC and vaterite as intermediates in the crystallisation of CaCO₃^21–25. However, in contrast to the unique nature of the equilibrium state, multiple reaction pathways from a given initial condition to that final state of thermodynamic equilibrium may exist. Such reaction pathways can be very sensitive to minor impurities and environmental perturbations, such as the presence of microorganisms, which modify the energy barrier from reactant to product phases²⁶. It has even been suggested that organic macromolecules associated with bacterial activity cause the Ostwald step sequence to stop at one of its intermediate stages: ACC → vaterite → calcite²¹, making biotic vaterite precipitation far more common than would be anticipated in abiotic systems. From a chemical standpoint, the polymorphous composition is governed by the addition of the reactants, which lead to a supersaturation state in which the concentration of calcium and carbonate ions exceed the solubility product of CaCO₃^18,27. This simultaneously triggers the nucleation of crystals and the dissolution of the colloidal ACC precursor¹⁸. However, local variations of the calcium and carbonate ion activity product (IAP) and thus saturation, can favour the formation of one polymorph over another. Vaterite, for example, typically precipitates in highly supersaturated and moderately alkaline environments^14,21,23.

Within this context, enzymatic hydrolysis of urea presents a straightforward process to understand the precise role of microbes in microbial induced calcium carbonate precipitation (MICP). This is because the urease enzyme is ubiquitous in microorganisms, yeast, and plants^5,28. In addition, it can be easily induced using inexpensive chemicals and is the most widely used process in biomediated soil improvement applications. Ureolytic bacteria enzymatically hydrolyse urea (CO(NH₂)₂), resulting in the production of ammonium (NH₄⁺) and dissolved inorganic carbon (DIC), which in turn increase pH and favour CaCO₃ precipitation in the presence of soluble calcium ions (eq. 1). This study focuses on three different ureolytic bacterial strains, all belonging to the Sporosarcina species: Sporosarcina pasteurii (ATCC 11859), Sporosarcina aquimarina (ATCC BAA-723), and Sporosarcina newyorkensis–a newly isolated microbe from the deep sea in offshore Japan extracted by the National Institute of Advanced Industrial Science and Technology (AIST) using pressure-core nondestructive analysis tools²⁹. To our knowledge, this has never been studied before. These strains were selected as they proliferate in different isolation environments: surface dry conditions, and shallow and deep sea, respectively.1aCO(NH2)2+2H2O→2NH4++CO32−\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{C}}{\rm{O}}({\rm{N}}{\rm{H}}}_{2}{)}_{2}+{2{\rm{H}}}_{2}{\rm{O}}\to {2{\rm{N}}{\rm{H}}}_{4}^{+}+{{\rm{C}}{\rm{O}}}_{3}^{2-}$$\end{document}1bCO32−+Ca2+→CaCO3↓\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{C}}{\rm{O}}}_{3}^{2-}+{{\rm{C}}{\rm{a}}}^{2+}\to {{\rm{C}}{\rm{a}}{\rm{C}}{\rm{O}}}_{3}\downarrow $$\end{document}

Our aim is to shed light on the interaction between the components of mineralised biological materials involved in CaCO₃ precipitation–namely minerals, macromolecules and water–, and understand their influence on the stabilisation of different CaCO₃ polymorphs. In addition, we wish to understand how strain-specific precipitation kinetics promote and affect this process. For this purpose, CaCO₃ was precipitated in vitro in 14 mL test tubes via the three different ureolytic soil bacteria described above. To ensure analogous reference conditions, strains were cultivated under sterile conditions at the same pressure and temperature–i.e. P = P_atm and T = 30 °C–to an optical density (OD₆₀₀) of ~0.5, and subsequently subject to identical external treatment conditions. The cementation treatment liquid medium consisted of a premixed solution of an equimolar amount of urea and CaCl₂ (0.3 M), in addition to 3 gL⁻¹ of Nutrient Broth, all dissolved in deionised water. All samples were created using a ratio of bacteria solution to cementation solution of 1:2. Results strongly suggest that the presence of structural water together with specific amino acids is fundamental to the stabilisation of vaterite and that, at the same initial OD₆₀₀ and treatment conditions, different strains yield distinctly different polymorphs. For this reason, we compared the precipitation kinetics, and the pressure-temperature dependence of bacterial population and urease activity for the three microorganisms. Finally, S. pasteurii–which is the most common soil bacterium used in geotechnical engineering applications–was also investigated under varying urea-CaCl₂ solution concentrations and initial OD₆₀₀. The mineralogy, morphology, and properties of precipitates were characterised using an array of complementary techniques, namely thermogravimetric analysis coupled with mass spectroscopy (TGA-MS), Raman spectroscopy (RM), X-ray powder diffraction (XRD), and scanning electron microscopy (SEM); and the precipitation kinetics of the three microorganisms quantified through measurement of calcium ion (Ca²⁺) concentrations and pH (Table S4). Ultimately, our results suggest that strain-specific CaCO₃ precipitation occurs during ureolytic MICP, possibly due to differences in the urease enzyme, and its response to treatment concentrations and pressure-temperature variations, and that CaCO₃ polymorphism in biotic systems is far more common than previously anticipated. This may have significant implications for biomediated soil improvement systems.

S. aquimarina induced the formation of highly porous rounded polycrystals, which had no well defined structure. Raman spectra collected for a single crystal up to a penetration depth of 25 μm showed a transition from calcite to vaterite with depth, evidenced by the splitting of the v₁ mode into two peaks (Fig. 2a). To our knowledge, such Raman characterisation of CaCO₃ polymorphs has never before been carried out and provides further evidence that biotic calcite initiates from a metastable phase rather than from a crystalline nucleus leading to single crystals. Moreover, hollow cores with walls formed by a concentric channel-like structure of interconnected pores were observed by SEM and suggested radial growth⁴⁹. While some cores still showed traces of vaterite spherulites, others had already started to be filled by advancing crystallisation steps (Fig. 2b). This morphology further reinforced the existence of a metastable precursor, which adopts a spherical structure to minimise its surface contact with the surroundings¹⁵, and provided significant evidence of a vaterite-calcite transformation.

Furthermore, our results indirectly showed that vaterite only stabilised when water molecules and amino acids were present in the crystal structure–i.e. SA01-0.3M, and SP02-0.2M-D and SP03-0.2M-ND. The idea that organic macromolecules can stabilise vaterite has been suggested previously, both in biotic¹⁴ and abiotic^50,51 systems. However, their presence alone as a justification for stabilisation seems unlikely as precipitates of S. newyorkensis only showed calcite peaks despite containing amino acids within their structure (Fig. 1a and Fig. S1). According to our TGA data, precipitates containing vaterite also comprised of half a molecule of water per one molecule of CaCO₃. Therefore, global rationalisation of these results was possible by considering water molecules, potentially from small amounts of additional hydrated phases that become structurally interrelated with the crystal structure. This likely happens via a hydrogen bond between the amino acid surface along the edge of the crystal and the water molecule. This scenario was supported by the different composition of amino acids in precipitates of S. newyorkensis and S. aquimarina (see supplementary discussion on pyrolysis of amino acids). Hence, our results, as well as those of other studies^52,53, suggest that only specific amino acid groups interact with the CaCO₃ surface, thus having an effect on the properties of the precipitated phases. In particular, polar hydrophylic amino acids–i.e. those amino acids that have oxygen and nitrogen atoms and with an unequal distribution of electrons–would be the only ones able to form hydrogen bonds with water molecules, either as proton donors or acceptors. The presence of nitrogen during the pyrolysis of precipitates of S. aquimarina lent some support to the existence of these hydrophilic amino acids within the crystal structure.

To better understand the underlying mechanisms during strain-specific ureolytic precipitation of CaCO₃, the precipitation kinetics were quantified and compared. Results showed that the precipitation (or not) of ACC or vaterite as precursors and/or intermediates of calcite could be favoured by selecting microorganisms with appropriate urease activities, and therefore, precipitation kinetics (cf. Fig. 4 and Table S4). S. pasteurii, the low-urease activity microorganism, had the fastest initial nucleation kinetics, and S. newyorkensis, the high-urease activity microorganism, had the slowest initial nucleation kinetics. Subsequently, crystallisation pathways of CaCO₃ polymorphs appeared to be highly pH- and dissolution rate-dependant. High pH and longer dissolution times following a fast nucleation event prompted ACC stabilisation (SP01-0.3M); while a pH closer to neutral and shorter dissolution times following a slower nucleation event promoted an ACC-calcite transformation (SN01-0.3M). Values for S. aquimarina (SA01-0.3M), yielding an ACC-vaterite-calcite transformation, were in between. Moreover, regardless of the formation pathway, CaCO₃ polymorph transformation involved the release of water (and the associated pH drop). Therefore, these results provide additional evidence that controlling dissolution kinetics is essential to controlling polymorph stabilisation. Nonetheless, establishing the specific mechanism via which this occurs requires direct amino acid analysis.

Because the kinetics of mineralisation were found to depend on (specific) urease activity, we sought to understand the role of cultivation conditions. Findings suggest that OD₆₀₀ is most sensitive to temperature, whilst (specific) urease activity displayed a more complex pressure-temperature dependence. The increase in OD₆₀₀ with temperature (Fig. S5a), most notable for S. newyorkensis and S. aquimarina, may, to some extent, be attributed to the decrease in dissolved oxygen contents. Indeed, an oxygen-limited environment would have prompted the growth of (facultative) anaerobic microorganisms. There is however a large body of research supporting the idea that pressure and temperature do not only affect the microbial ecology of aqueous environments, but also the structural stability of biomolecules^54–56, the cell membrane composition⁵⁷, and the physiology of bacterial cells⁵⁸. Therefore, further work is required to assess the role of pressure and temperature on these processes, as well as on how they affect the preferred CaCO₃ phase precipitated by the bacterial strains studied here.

Results showed that optimum conditions for CaCO₃ polymorphism are both strain- and environment-dependant (i.e. urea-CaCl₂ solution, pressure, temperature). Under the same bacterial concentrations and environmental conditions the three bacterial strains studied herein produced different polymorphs (Fig. 1a), most likely due to the differences in their urea hydrolysis metabolism process. In addition, the results for S. pasteurii showed a coupling between the concentration of ions in solution (i.e. supersaturation) and the initial bacterial concentrations. Results suggested that the higher the ionic strength, the stronger the interaction between background ions and water becomes. This reduced the availability of water molecules in the solution, favouring clustering and dehydration, and thus precipitation. Conversely, at low ionic strengths, dehydration was hindered due to the higher availability of water molecules in the solution⁶. This promoted hydrophilic interactions between amino acid and water molecules, affecting the dissolution-reprecipitation via which vaterite transforms into calcite. It is worth pointing out that in biotic systems, high ionic strength is attained through the combination of high bacterial concentrations–i.e. yielding high carbonate ion concentrations–and high urea-CaCl₂ solution concentrations, and it is this trade-off between the two that will determine whether metastable polymorphs stabilise or not.

As a general rule, calcite has been the only CaCO₃ polymorph reported for geotechnical engineering applications, as this is thought to be less prone to alteration, i.e. more stable. However, this study supported the notion that ACC and vaterite are much more abundant in CaCO₃ biomineralisation than previously believed, and clarifying how they are formed and stabilised may have broader implications for the mechanical properties of cemented sands. Generally speaking, the role of MICP in sands is to create an adhesive bond at the inter-particle contacts, enhancing their load-carrying capacity. In this context, particle contact properties will both be affected by the degree of cementation and the nature of the cementing bond (e.g. morphology, mineralogy, properties). Both numerical and experimental data have shown that increasing the cementation level enhances the maximum shear strength and stiffness at small strains, and as the cementation increases the stress-strain behaviour transitions from ductile to brittle^9,59,60. From an engineering standpoint, this leads to an unwanted soil response because brittle materials absorb little plastic energy prior to fracture, and thus fail catastrophically. Clearly, it is also noted that different CaCO₃ polymorphs yield different particle contact properties, leading to variations in their strengthening effect. DEM analyses in the literature have shown that an increase in inter-particle friction brings the stress-strain response of granular materials from ductile to brittle, and augments the anisotropic distribution of contact forces⁶¹. For the specific case of MICP-treated soils, studies further concluded that increasing calcite content results in a few heavily loaded particle contacts transmitting a large proportion of the load, thus making the force distributions become increasingly non-uniform⁵⁹. This is consistent with the fact that higher friction values tend to decrease the mean contact number per particle⁶¹.

Of key importance therefore, is that the CaCO₃ polymorph selection may be controlled through selection of a bacterial strain with appropriate precipitation kinetics, while metastable polymorph stabilisation may be controlled by inhibiting dissolution through the interplay between specific amino acid groups and water within the crystal structure. Further, this study showed that ureolytic microorganisms common to geotechnical engineering environments not only adapt to the urea-CaCl₂ solution by modulating the precipitation kinetics, but that this interaction is very sensitive to pressure and temperature. In particular, the increased sensitivity of urease activity to pressure at temperatures near those of the original isolation environment is of major importance for the application of MICP in the deep sea, where both high pressure and low temperature combine to produce a highly hostile environment for bacterial viability.

Results for S. pasteurii further revealed that biotic precipitation of calcite alone required both high urea-CaCl₂ solution concentrations and high bacterial concentrations, treatment conditions that would yield substantial amounts of ammonium ions (NH⁴⁺), a hazardous byproduct of urea hydrolysis. However, using a bacterial strain that is able to produce the desired polymorph at lower bacterial and treatment concentrations (e.g. S. newyorkensis) offers potential to lower the yield of this byproduct. Therefore, an optimisation exercise between bacterial and treatment concentrations is required on a strain-specific basis for biotechnological applications.