Bacillus megaterium favors CO₂ mineralization into CaCO₃ by ureolytic pathway

In this work, we aim to investigate a less explored microorganism in the traditional field of microbially induced calcite precipitation (MICP), which is dominated by ureolytic strains^1,2,3,4. To understand the motivation behind this work, one should consider that the traditional MICP pathway involves the breakdown of urea (an organic fertilizer produced via the combination of liquid CO₂ and liquid ammonia) by urease-producing microorganisms such as Sporosarcina spp. into CO₃²⁻ and NH₄⁺ (Eq. 1). The ammonium ion is an unwanted byproduct that must be completely removed as a trade-off in the otherwise beneficial MICP process, in which CaCO₃ precipitate acts as a binder for building applications, commonly referred to as biocement (Eq. 2)⁵.

In addition to traditional ureolytic MICP, among the different metabolic pathways that facilitate carbonate precipitation, the use of carbonic anhydrase (CA) is attracting interest. In recent works, microbes with genetic information for the CA enzyme have been mobilized in an attempt to eliminate ammonium, for example, in the work of⁶, in which Bacillus licheniformis was used in bioaugmentation protocols to improve clay structure. In this method, the applied MICP pathway emerged from a preliminary step in which hydrated aqueous CO₂ formed H₂CO₃ upon reaction with water and subsequently ionized, yielding HCO₃⁻ and water (Eq. 3). The newly ionized carbonate anion then combined with free Ca²⁺ cations to form CaCO₃ in one of its metastable phases, i.e., into the amorphous calcium carbonates (ACCs) vaterite or aragonite or to directly form calcite (Eq. 2). CA is known to serve as a nucleation site in this process^6,7.

We therefore focused on Bacillus megaterium, a species with genetic determinants for both the enzymes urease and carbonic anhydrase. These two pathways, in fact, can be activated at the same time synergistically⁸, but it is unclear whether selective activation can occur. Mineralization of gaseous CO₂ by B. megaterium has been reported in⁹, and given increasing interest in the direct use of atmospheric CO₂ in industrial processes, in this study, we suggest a framework for tracing the carbon source of precipitated CaCO₃ and quantifying the fraction that originates from CO₂ (Fig. 1).

Fig. 1

Illustration of urease and CA-induced MICP in B. megaterium using urea and CO₂ as reactants, respectively.

Bacillus megaterium is an aerobic gram-positive bacterium commonly present in soils¹⁰. It received its unique name from its dimensions: 4 μm long and diameter of 1.5 μm¹¹. The interest in this taxon lies in its versatility; it has been applied for recombinant protein production¹², soil bioremediation¹³, and microbially induced precipitation¹⁴. The enzyme urease catalyses the reaction, and one mole of urea is hydrolysed to ammonia and carbonic acid¹⁵. This leads to an increase in pH and the number of negative ions on the cell surface, which attract positively charged calcium ions, leading to CaCO₃ precipitation. B. megaterium has the ability to exploit another pathway to induce carbonate precipitation¹⁰ via CA-mediated precipitation. An alkaline environment induces a reaction between calcium ions and HCO₃⁻ and ultimately leads to calcium carbonate precipitation. Nevertheless, environmental conditions, such as pH, temperature¹⁶ and the presence of ions like magnesium^17,18, have a complex and significant effect on the precipitation of CaCO₃ polymorphs, influencing not only the crystallization process but also the morphology and stability of the resulting phases. Despite this, the selective activation of ureolytic metabolism as a mechanism for controlling CaCO₃ polymorphism remains less explored, warranting further investigation to better understand its role in microbial-induced calcium carbonate precipitation.

CaCO₃ precipitation via the urease pathway

To confirm the urea hydrolysis pathway in MICP, a first round of growth experiments (U test) was performed in aerobic 100 mL flasks on the basis of conditions proposed by¹⁹. B. megaterium (DSM 32) was grown in 15 g/L yeast extract, 10 g/L peptone, and 10 g/L NaCl (growth medium) for 24 h. All the media components were obtained from Sigma Aldrich unless otherwise indicated. Subsequently, 4.5 mL of B. megaterium culture was inoculated (OD₆₀₀ = 0.7) in 30 mL of media supplemented with urea and CaCl₂. Both components were filtered through 0.22 μm filters (Sigma‒Aldrich) and added in equimolar proportions at 1.5 M. The flasks were incubated in triplicate for four days at 30 °C and 100 rpm. Daily feedings of the growth medium were performed, and pH was monitored at the end of each experiment together with precipitate yield, absorbance and electrical conductivity (see Supplementary Figure S1). One should consider herein that alkaline environments can be linked to the presence of peptone itself (10 g/L), which may influence the pH of the medium, or potentially magnesium, which is known to preferentially precipitate aragonite²⁰. However, magnesium was not used in this study. Furthermore, abiotic control samples, including peptone, were tested in this work and yielded no precipitation, supporting the notion that the presence of peptone alone does not significantly impact the absorption or hydration of CO₂ in the culture medium. Data from these abiotic tests are provided in the Supplementary Figure S1. In CO₂-infused media, suboptimal pH conditions for CaCO₃ precipitation are expected due to acidification. While such environments are often considered unfavorable for biomineralization, recent studies show that B. megaterium retains urease activity and supports calcite formation even below neutral pH. More precisely²¹ observed measurable growth and precipitation at pH 6–7, and Manna et al. (2024)²² confirmed these findings across a broader acidic-to-alkaline range. These results indicate that CaCO₃ precipitation cannot be excluded under mildly acidic, CO₂-rich conditions. Additionally, we postulate that peptone concentrations around 10 g/L are typically sufficient to support bacterial growth and promote ammonia production from urea (which was supplied at 90 g/L), thus contributing to medium alkalinization, as described by²³. These starting hypotheses highlight how these factors—alkaline environment, peptone, and the absence of magnesium—contribute to the formation of CaCO₃ polymorphs via enzymatic pathways in the proposed experimental system.

CaCO₃ precipitation via the carbonic anhydrase pathway

The second round of experiments (CA test) was designed to evaluate CaCO₃ formation via the carbonic anhydrase pathway. For this purpose, 2.5 mL of B. megaterium grown for 24 h was inoculated (OD₆₀₀ = 0.7) in fresh medium supplemented with C¹³-labelled urea (Sigma‒Aldrich, CAS 58069-82-2), and CaCl₂ was added in an equimolar amount at 1.5 M (Table 1). This experiment was conducted in closed vials in triplicate. Thirty millilitres of CO_2, corresponding to 1.24 × 10⁻³, was injected into each vial via a 30 mL syringe. The samples were incubated for four days at 30 °C and 100 rpm. Daily feedings of the growth medium were performed, and pH was corrected to above a value of 8. The precipitate in this test was used to determine the origins of the carbon in the formed CaCO₃.

The available CO₂ in the liquid phase was calculated on the basis of Henry’s law considering Henry’s law constants and the gas partial pressure²⁴.

Precipitate analysis

The collected samples were centrifuged for 4 min at 624 × g, and the pellet was washed with acetone (Sigma Aldrich, 99% purity). After the pellet was dried overnight at 60 °C, a second washing step with 96% ethanol was performed. The samples were subsequently dried and heated at 450 °C for two hours before Fourier transform infrared (FTIR) spectroscopy (Spectrum two, Perkin Elmer) and scanning electron microscopy (SEM) analyses with energy dispersive X-ray spectroscopy (EDX) (Phenom XL, Thermo Scientific) were performed. The organic and inorganic origins of the CaCO₃ precipitate was investigated by analysing the C¹³ -labelled fraction of carbonate via the double-capsule technique described in²⁰.

CaCO₃ production in the U test

The FTIR spectra of the precipitates from the U test are reported in Fig. 2. The yellow line represents the calcite spectrum acquired from a reference database²⁵. The wavelength range between 400 and 1500 nm contains characteristic peaks for calcite: the absorptions at 712 cm⁻¹, 872 cm⁻¹, 1082 cm⁻¹, and 1394 cm⁻¹ are influenced by the different ways in which C-O bonds form carbonate²⁶. The black dotted line corresponds to the precipitate sample from the U test, and as expected, the characteristic peaks at 712 cm⁻¹, 872 cm⁻¹, and 1394 cm⁻¹ are identified and in agreement with the reference spectrum. Additionally, in the spectrum at approximately 1082 cm⁻¹, it is possible to distinguish a wider peak that spans several peaks. We postulate that this is due to the trace amount of other inorganic material that was still present in the pellet despite the washing procedure.

Fig. 2

U FTIR spectra of the calcium carbonate precipitate peaks compared with those of pure calcium carbonate peaks.

CaCO₃ production in the CA test

The FTIR spectra of carbonate from CA test are reported in Figure 3B. Similar to that in the previous experiment, the precipitate was identified as CaCO₃ by EDX (see Supplementary Figure, S1). To elucidate the polymorphic phase of CaCO₃, the spectra were subsequently analysed and compared: calcite and aragonite data were collected from the reference database (Perkin Elmer Spectrum IR), whereas the spectra of vaterite were taken from the work of²⁷ (Figure 3A). As mentioned above, calcium carbonate has three characteristic IR absorption peaks in the range between 400 cm ^{and 1} and 1500 cm⁻¹ due to the interaction of the C-O bonds with infrared radiation. CaCO₃, in fact, has three crystalline structures, all with the same chemical composition: calcite, aragonite, vaterite, and each crystalline structure can be associated with a corresponding spectrum (Figure 3A). A comparison of the calcite and aragonite FTIR spectra (Figure 3A) revealed that in the aragonite absorption spectra, the peak at 712 cm⁻¹ is shifted, with an additional peak at 700 cm⁻¹; this peak is not present in the calcite phase. In both the vaterite and calcite spectra (Figure 3A), the peak at 875 cm⁻¹ is matched; on the other hand, the peak at 712 cm⁻¹ present in calcite is shifted to 748 cm⁻¹ for vaterite²⁸. Furthermore, the third characteristic peak of calcite (at 1082 cm⁻¹) is slightly shifted in the vaterite sample. In the CA test, the obtained spectra are consistent with the peaks corresponding to calcite (Figure 3A), which confirms that B. megaterium-induced MICP leads to the formation of calcite²⁷.

Fig. 3

(A) FTIR spectra of calcite, aragonite and vaterite; (B) CA test results compared with those of calcite.

In addition to FTIR analysis, using the pellet collected from the CA Test, the textural and morphological characteristics of the crystals formed by B. megaterium were investigated using SEM. In the samples collected from the fresh medium (Figure 4A) and from the double-washed, dried pellet (Figure 4B), no dominant morphological or textural features were identified, and B. megaterium cells could not be distinguished. This could be explained by the predominant distribution of the microorganisms in the supernatant rather than in the pellet collected from the bottom of the flask after centrifugation. In the washed and dried precipitate (Figure 4B), as expected, no microbial cells were identified, but a clearer and more hierarchical matrix was observed for the residual crystals that resisted the washing and drying process. More precisely, the inorganic fraction had different morphological structures, i.e., rhombohedral, spherical, and amorphous. We postulate that the applied protocols resulted in the coexistence of various polymorphs of calcium carbonate, as observed in other MICP studies¹⁴. Therefore, it can be concluded that the final polymorph of calcium carbonate formed depends on the amount of dissolved organic carbon (DOC) present. When DOC is present in low quantities, calcite is more likely to form, whereas when DOC is present in high quantities, the formation of vaterite is favoured instead⁸.

Fig. 4

CA test scanning electron microscopic observations at magnifications of 3000x, 5000x, 8000x at 15 kV and 10 Pa; (A) Pellet collected from fresh samples; (B) pellet postdrying and postwashing.

The results from duplicate analyses using the double-capsule technique²⁰ allowed for the quantification of the fraction of CaCO₃ that contained the C¹³-labelled organic precursor (urea). Notably, 94.1% (mean value, with a standard deviation of ± 0.1%) of the total carbon was unlabelled, which implied that the vast majority of the precipitated CaCO₃ was formed from the incorporation of inorganic carbon, i.e., CO₂ was identified as the main carbon source and precursor of the mineral phase. This result highlights that in the presence of increased concentrations of CO₂ (1.24 × 10⁻³), B. megaterium tends to exploit its carbonic anhydrase metabolic pathway predominantly instead of the ureolytic pathway for the synthesis of CaCO₃.

References

Authors and Affiliations

1. Environmental Biotechnology, Institute of Microbiology, Department of Environment, Construction and Design, University of Applied Sciences and Arts of Southern Switzerland (SUPSI), Manno, Switzerland

   Margherita Cappa, Camilla Perego & Pamela Principi

2. Swiss Federal Institute of Technology, Lausanne (EPFL), Faculty of Environment, Architecture and Civil Engineering (ENAC), Lausanne, Switzerland

   Dimitrios Terzis

3. Medusoil SA, EPFL Innovation Park Building A, 1015, Lausanne, Switzerland

   Dimitrios Terzis

Contributions

All the authors contributed to the design and execution of the experiments presented in this manuscript, as well as to the interpretation of the results. Each author reviewed and approved the final manuscript prior to submission.

Corresponding author

Correspondence to Dimitrios Terzis.

Competing interests

The authors declare that there are no competing interests related to this publication. The research, analysis, and findings presented are independent of any financial, professional, or personal relationships that could influence the content or interpretation of the results.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions