Amorphous and crystalline CaCO3 polymorphs

XRD analysis (Fig. 1a) revealed that calcite was the primary polymorph that precipitated in the presence of S. newyorkensis (SN01-0.3M), along with small traces of halite resulting from the drying of the marine broth media used to cultivate the isolate30 (Fig. S1). On the other hand, precipitates of S. aquimarina (SA01-0.3M) contained vaterite as a secondary phase to calcite. The XRD spectrum of precipitates of S. pasteurii (SP01-0.3M) showed no traces of calcite, but a broad hump in the range of 15–40° 2Θ consistent with the presence of a poorly-ordered material and matching data reported in the literature for ACC31,32.

Figure 1

XRD pattern with hkl values of Bragg peaks indicated (Cu-Kα radiation, λ = 1.5406 Å) (a); and TG curves (All: heating rate 10 °C min1; SN01-0.3M: Ar “reactive gas” flow rate 50 mL min−1, and SA01-0.3M and SP01-0.3M: N2 “reactive gas” flow rate 50 mL min−1 (b) of precipitated CaCO3. V, vaterite; Cc, calcite; Halite peaks resulting from desiccating media of S. newyorkensis not indicated.

Thermal analysis of the same samples also showed significant differences (Fig. 1b). S. newyorkensis was characterised by the occurrence of three main weight loss steps, at 217 °C, 438 °C, and 757 °C, corresponding to 2.4, 9.1, and 38.1 wt%, respectively. In contrast, the TGA of precipitates of S. aquimarina only showed two main weight loss steps at approximately 300 °C and 717 °C. However, the first derivative (DTG) revealed that the former resulted from the overlap of four peaks at 184 °C, 251 °C, 287 °C, and 348 °C (Fig. S2). These values appeared too high to be attributed to physisorbed water–i.e. water evaporated below 115 °C19–and were thus attributed to weakly (~30–200 °C) and strongly (~200–550 °C) bound water molecules, giving a structural water content of 8.0 wt% from TGA33. Furthermore, the large weight loss observed for S. newyorkensis and S. aquimarina at 757 °C and 717 °C (36.4 wt%), respectively, was consistent with the loss of CO2 from the carbonate decomposition, and also provided an independent confirmation that both samples were comprised almost solely of CaCO3 phases. Regarding the precipitates of S. pasteurii, TGA revealed that below 250 °C there were two distinguishable temperature intervals where weight losses occurred, namely at 112 °C and 231 °C. The total weight loss in these transitions was 27 wt% (after physisorbed water removal) and was attributed to dehydration and crystallisation of ACC34. The third weight loss was 18 wt% and occurred in the temperature range of 300–550 °C. At such high temperatures, this was unlikely to be caused by the release of structural water, and was thus associated with the pyrolysis of macromolecules, either of organic or inorganic origin. Finally, the thermal peak at 670 °C, matching the decarboxylation of CaCO3, only accounted for 8 wt% of the weight loss, indicating that minor amounts of CaCO3 were present. The final plateau, slightly inclined, indicated that final weight constancy was not achieved in this sample, possibly due to kinetic effects upon carbonate decomposition33.

Biotic vaterite precipitation

To further investigate how vaterite and calcite were spatially organised, the precipitates of S. aquimarina were examined through Raman spectroscopy. Firstly, spectra were acquired at two visually distinct points of a single crystal. Results are shown in Fig. S3, together with an optical microscopy image of the target collection points. The most prominent features of calcite are the symmetric stretching mode (v1) of the carbonate group, followed by two external modes35,36. These appeared at 1085 cm−1 with a full width at half maximum (FWHM) of 7 cm−1, and at 155 and 280 cm−1, respectively. The identification of other features was not possible due to the high background noise. Raman spectroscopy also helped distinguish vaterite from calcite by comparing the wave numbers of the v1 mode. Indeed, the absorption bands at 1076 and 1088 cm−1 corresponding to the symmetric stretching of vaterite37 were detected in one of the spectra shown in Fig. S3. Secondly, spectra of a single crystal were also collected up to a penetration depth of 25 μm, with results clearly showing a polymorph transition (Fig. 2a). While the characteristic single peak of calcite at 1086 cm−1 was detected at the surface, the two peaks at 1076 cm−1 and 1088 cm−1 appeared within the internal structure. Moreover, the broad nature of the vaterite peak at 1088 cm−1 could possibly suggest the combination of two peaks at 1090 and 1085 cm−1, the latter being associated to calcite. This is due to the peak convolution between the polymorphs in this region. In addition, the disappearance of the two lower frequency lattice modes (155 and 280 cm−1) with penetration depth further supported a transition between carbonate phases. Table S1 lists the peak positions and the corresponding assignments of calcite and vaterite to illustrate the comparison between precipitates of S. newyorkensis and S. aquimarina.

Figure 2

Raman spectra of polymorphic CaCO3 crystals formed in the presence of S. aquimarina (SA01-0.3M) evidencing the coexistence of calcite and vaterite within a single crystal and showing their spatial arrangement (a); and SEM BSE image of internal structure of biotic precipitates of S. aquimarina with some cores showing traces of vaterite spherulites (yellow dotted circle) and others that have started to be filled by advancing crystallisation steps (red solid circle) (b).

SEM images of precipitates of S. aquimarina showed two distinctive morphologies: (a) spherulites, 5–50 μm in diameter (Fig. S4a), which were associated to vaterite and disphenoid- and dipyramid-like calcite crystals (Fig. S4e). Numerous rod-shaped bacterial cells (with length ~2 μm and a diameter of ~0.5 μm) were observed encased within the growing spherulites, suggesting that the presence of S. aquimarina was a prerequisite for their formation. This was further reinforced by the presence of smooth dumbbell CaCO3 morphologies, which have been reported to be uniquely bacterial in origin (Fig. S4b)38. In addition, an epoxy cast cross-section of the precipitates (Fig. 2b) revealed hollow cores with walls formed by a fibro-radial internal structure, an observation consistent with spherulite surface features described by39. While some cores still showed traces of vaterite spherulites, others had started to be filled by advancing crystallisation steps. Further key aspects of the incorporation of the vaterite spherulites into the calcite crystals were obtained by comparing Fig. S4b–e.

Indirect evidence for structural amino acids and water

As evidenced by Fig. S8a-b, the XRD and Raman spectroscopy of precipitates of S. aquimarina and S. newyorkensis also showed the presence of additional absorption bands that did not correspond to carbonate phases. In particular, the Raman spectra showed the Disorder (D) and Graphite (G) bands typical for organic carbons, with those present in precipitates of S. newyorkensis (SN01-0.3M) exhibiting a slightly lower level of organisation than those observed within preciptates of S. aquimarina (SA01-0.3M) (see zoomed in plot Fig. S8b and associated supplementary discussion, and Table S2). Consequently, TG-MS analysis on two different powdered samples was used to identify and monitor the evolution of the exhaust gases. MS was set to detect certain m/z values associated with common fragments from molecular-ions, listed in Table S3. All MS signals were normalised to a baseline shift value obtained after each experiment from a blank run using an empty crucible. Results therefore refer to relative incremental yields rather than absolute intensity values per se. The advantage of this was to be able to compare all exhaust gases using a single plot, allowing for thermal decomposition sequences to be unequivocally identified. The only exception was CO2, plotted on its own intensity axis because its relative yield was considerably higher than that of the other products–understandable given that all of the analysed precipitates were carbonates. Therefore, where discussion warranted, zoomed in plots of the range of temperatures of interest showing the CO2 yield with respect to the other products in the group were presented.

The TGA of precipitates of S. newyorkensis showed the occurrence of three main weight loss steps. As shown in Fig. 3a, CO2 was the main gaseous product in the third step (757 °C), associated with the decarbonation of CaCO3. Regarding the first and second steps (217 and 438 °C), decomposition products mainly included NH3 and H2O, respectively. This indirectly demonstrated the presence of amino acids within the precipitated carbonates and suggested that their primary decomposition included deamination with low yields of dehydration. Using Fourier transform infrared spectroscopy (FTIR), previous studies identified the presence of amino acids within the CaCO3 structure of biotic precipitates by the amide I signature at 1655 cm−114,40,41. Further, FTIR for evolved gas analysis coupled to TGA revealed that the thermal decomposition of organics results in the release of CO, CO2 and NO2 in the temperature range of 150–500 °C, while amino acids in biotic carbonates also involve the release of NH314. These results are in agreement with the TG-MS analyses reported here. Moreover, Fig. 3a shows that the rapid releasing rate of NH3 in the first stage was in sharp contrast with the longer H2O and NH3 releases observed in both the second and third stages respectively, revealing different pathways of formation. In the former, NH3 was most likely lost as a result of a primary decomposition (i.e. individual molecular decomposition of amino acids or formation of an amino radical), while H2O in the second stage may have been produced following secondary reactions42,43. One interesting observation was that CO2 was not released during the first stage (see zoomed in plot of Fig. 3a), suggesting that less common aromatic β-amino acids were present42 (Fig. S8b).

Figure 3

DTG and mass spectra of evolved gases measured from coupled TG-MS of CaCO3 crystals formed in the presence of: (a) S. newyorkensis (heating rate 10 °C min−1; Ar “reactive gas” flow rate 50 mL min−1); and (b) S. aquimarina (heating rate 10 °C min−1; N2 “reactive gas” flow rate 50 mL min−1).

Regarding precipitates of S. aquimarina, their pyrolysis was markedly different (Fig. 3b), with the first two decompositions being less sharp and partially overlapping. The most conspicuous feature was that water remained a structural constituent up to 600 °C, gradually being released from 100 °C (8 wt%). Additionally, the fragmentation products and sequence of precipitates indirectly suggested different starting amino acids, with deamination being a primary, although minor, mode of decomposition. This was echoed by the small NH3 peak measured between 100–160 °C, previously observed during the pyrolysis of α-amino acids and attributed to the existence of an intermediate44. On the other hand, a second process, also considered to be a primary decomposition mode, was the decarboxylation reaction of α-amino acids to produce CO2 and amines42,44. This was evidenced by the CO2 peak measured at 243 °C (see zoomed in plot of Fig. 3b). Following this primary decomposition, a number of secondary products arise, possibly from the fragmentation of the amines themselves (see supplementary discussion for further details on the pyrolysis of amino acids).

CaCO3 precipitation kinetics

With approximately equal OD600 ~ 0.5 but different (specific) urease activities (Table S4), it is suggested that the mineralogy, morphology and properties of the precipitated CaCO3 can be controlled by ureolytic strains with different urease activities and, consequently, precipitation kinetics–quantified through measurement of calcium ion (Ca2+) concentrations and pH45.

Equal concentrations of Ca2+ ions and bacterial densities were initially present in all tests, with CO32− ion concentrations being equal to zero until the onset of urea hydrolysis. As a result, initial calcium depletion rate–calculated as the change in Ca2+ ion concentration over a certain period of time, dCa2+/dt–was most closely associated with the nucleation of CaCO3 for each microorganism45. A lower initial urease activity was associated with a faster initial calcium depletion rate from solution (cf. Table S4 and Fig. 4a). Comparing the results for S. pasteurii (SP01-0.3M) with those for S. newyorkensis (SN01-0.3M) and S. aquimarina (SA01-0.3M) it is clear that S. pasteurii’s metabolic activity was able to initially hydrolyse more urea into CO32− ions in the same period of time. This resulted in alkalinity generation and ammonia release, which respectively caused a rapid increase in supersaturation and pH. This provided the high crystallisation kinetics needed for the spherulitic precipitation of ACC, which proceeds via a fast nucleation-controlled mechanism46–again evidenced by the higher initial calcium depletion rate. Further, the ACC-vaterite transformation in precipitates of S. aquimarina manifested by a slow decrease in pH associated with the release of water molecules during dissolution; and by a decreasing Ca2+ depletion rate associated with the release of Ca2+ ions stored in ACC and their re-precipitation into vaterite. Conversely, this dissolution (and associated pH drop) was not observed in SP01-0.3M, further reinforcing the stabilisation of ACC.

Figure 4

Time evolution of the (a) rate of calcium depletion and (b) pH.

The initial Ca2+ depletion rate was smallest for S. newyorkensis, indicating a slower nucleation event. Interestingly, this reversed as Ca2+ ions returned to solution, suggesting the early dissolution of Ca-containing precipitates (most likely ACC). As with the initial calcium depletion rate, the timing of this transition appeared to depend on the microorganism. For S. newyorkensis, with the highest urease activity, this transition occurred earlier than for the other two less ureolytic microorganisms. Figure 4b further suggests that this process was highly pH-dependant. For S. newyorkensis (pH ≈ 7–7.5), calcium ions returned to solution within the first 24 h, and depleted again following a second nucleation event (calcite). This reinforced a direct ACC-calcite transformation when initial pH values are closer to neutral suggested in the literature46. With increased pH however, vaterite (pH ≈ 7.5–7.8) and ACC (pH ≈ 7.8–8.3) were stabilised by S. aquimarina and S. pasteurii, respectively. This was linked to a delayed return of Ca2+ to solution.

An additional consideration is that the pressure-temperature conditions in the environments where the three bacterial strains proliferate are substantially different, thus affecting bacterial growth and urease activity. These parameters were therefore investigated during a 4-day cultivation at two different pressures (1 atm, 3 MPa) and three different temperatures (4 °C, 15 °C, 30 °C). Interestingly, results showed that the effect of temperature on OD600 was more pronounced than that of pressure (Figs. S5a and S6a), and that (specific) urease activity was most sensitive to pressure at temperatures near those of the original isolation environment (Fig. S5b,c). Fig. S6b shows that a loss of specific urease activity with pressure for all three ureolytic microorganisms, suggesting that pressure potentiates inhibitory factors that affect the function of the urease enzyme. It is also clear that aside from pressure and temperature, time played an important role, with the following conditions yielding a maximum specific urease activity (all at P = 1 atm): S. newyorkensis, 1-day cultivation, T = 4 °C; S. aquimarina, 1-day cultivation, T = 15 °C; and S. pasteurii: 4-day cultivation, T = 30 °C.

Cooperative effect of bacterial and treatment concentrations

We further extended our study by investigating the influence of OD600 and urea-CaCl2 solution molarity on polymorphic selection for S. pasteurii (Fig. S7). For example, SP02-0.02M-D refers to S. pasteurii treated with 0.02 Molar of CaCl2 and with a diluted OD600 of 1.38. TGA showed that samples treated with 0.02 and 0.2 M displayed almost identical thermogram profiles between bacterial concentrations (Fig. S7a–d). Close examination of the 0.02 M samples (D and ND) disclosed that in both cases the amount of CaCO3 was ~15–30 wt%, which was found to be in agreement with values reported in other biotic studies of ACC15,47. Furthermore, two distinguishable temperature intervals where weight loss occurred were measured below 250 °C. These were accompanied by broad peaks at around 110 and 230 °C and were attributed to the release of water molecules. This double dehydration behaviour was also consistent with previous studies15,47,48. The total weight loss in this transition was 15–16 wt%, which was compatible with a stoichiometry of CaCO3·H2O. Finally, a third weight loss region of approximately 25 wt% occurred in the temperature range of 250–500 °C. At such high temperatures, this loss could be attributed to either strongly bound water molecules and/or to the secondary decomposition of amines (cf. Fig. 3a). The wider nature of this peak (i.e. slower release), however, lent some support to the existence of secondary reactions (see supplementary discussion on the pyrolysis of ACC and Fig. S12). Acquisition of XRD patterns for the 0.02 M samples was not possible due to the low volume of precipitates available. However, the similarities between the thermal decomposition observations made for these samples and SP01-0.3M, allowed us to conclude that XRD patterns would have been very similar to the one showed in Fig. 1a and attributed to ACC.

The TGA of precipitates resulting from the 0.2 M solutions (both D and ND) showed a clear peak at around 760 °C (~40 wt%), corresponding to the decomposition of CaCO3. In addition, their TG profiles were very similar to the profile obtained for precipitates of S. aquimarina (cf. Fig. 1b), with two small peaks at approximately 220 °C and 320 °C (8.2 wt%). Taken together, these results corroborated the presence of water within the crystal structure with a stoichiometry of CaCO3·1/2H2O19. In addition, the XRD measurements showed the presence of both vaterite and calcite, but no extra reflections attributed to crystalline hydrated phases were apparent (Fig. S7e,f).

Bacterial concentration had a greater effect at a solution concentration of 1 M, with calcite being the only phase detected at high OD600 (SP03-1M-ND Fig. S7f) and vaterite at low OD600 (SP02-1M-D Fig. S7e). The TGA of these samples provided further valuable insights into their properties. While SP03-1M-ND (Fig. S7b,d) showed a sharp peak at 240 °C–matching the first peak observed for S. newyorkensis (cf. Fig. 3a) and associated to the pyrolysis of amino acids–, the thermal decomposition of SP01-1M-D (Fig. S7a,c) started at a lower temperature (220 °C) and was accompanied by a second peak at 327 °C. In consistency with previous observations made for precipitates containing vaterite, and by comparing this profile with the MS results for precipitates of S. aquimarina (cf. Fig. 3b), results strongly suggested the presence of structural water and amino acids. Finally, both samples displayed a significant weight loss at 740 °C, corresponding to the decarbonation of CaCO3.

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