Characterization of the field collected ACC. Aethalia from 37 F. septica specimens were collected (See Field Observations under Methods) and stored under normal laboratory conditions. The aethalia consists of an inner spore mass and an outer peridium. The peridium is the thin, to 3-mm thick, brittle porous coating that covers and surrounds the spore-bearing aethalium (Figs. 1a, b, S1c). The specimens from southern Arizona are white (referred to as FSW for Fuligo Septica White) and the UK specimens are bright lemon yellow (referred to as FSY) (Figs. 1, S1, S2). The yellow of the FSY specimens is caused by a range of pigmented compounds including the tetramic acid derivative fuligorubin A 38, which also acts as a metal chelating agent 39,40. Observations of the Arizona specimens by the senior author showed formation of the aethalium during the evening and early hours of the morning within a few days following summer rains. By morning, the aethalia are fully developed and samples were collected for study. Fragments of the peridia free of the spores were separated for analysis (Fig. S2).
A combination of PIXE, TG analysis, and CHN analysis were used to analyze the elemental composition of the FSY and FSW peridia (Tables 1, S1, S2). Calcium is the major cation. The FSY samples contain ~3.3 wt% Mn, whereas the FSW samples contain between 937 and 2186 ppm Mn. Magnesium was typically at or below the PIXE detection limit of ~200 ppm: the only other elements consistently above the limit of detection for the PIXE are P, S, Cl, and K (Table S1).
Optical, SEM, and TEM images show the peridia composed of 500- to 1500-nm-sized spheres (Figs. 1, 2, S1d), consistent with previous SEM images 28. The spheres are isotropic under crossed polarized transmitted light (Fig. 1f). The high-angle annular dark-field scanning TEM (HAADF-STEM) and bright-field TEM (BFTEM) images show that the spheres typically have an internally mottled appearance. This mottling is especially evident in the BFTEM images, which show rounded, 20- to 40-nm-sized electron dense units (Figs. 2c and S3). The selected-area electron diffraction (SAED) patterns of the spheres show diffuse rings (Fig. 2d), similar to what was reported by Enyedi et al. 19 for bacterially precipitated ACC.
Multiple peridial samples were examined by powder XRD: all lack the sharp intense Bragg reflections of crystalline phases, and are instead dominated by five broad, but well-defined maxima at ~1.9, 0.46, 0.2866, 0.204 nm, 0.119 nm, and weak maxima at higher d-spacings (Fig. 3). The high signal-to-noise ratio of our powder XRD patterns, compared with published data, was possible because of the large amount of pure HACC produced by the slime mold, and its exceptional stability in air. For example, the XRD patterns of the FSW and FSY samples acquired within hours of formation compared with those stored in the laboratory for two years are identical. Except for the ~1.9 nm reflection, the diffraction patterns are similar to those for synthetic and biogenic ACC 5,7,26,41−43. The prominent ~1.9 nm has not previously been reported and suggests ordering at the ~2 nm scale.
The FTIR spectra of the HACC (Figs. 4, S4, S5) show the characteristic bands of ACC, with spectra similar in shape to biogenic (e.g., Fig. 2a and b, and in Addadi et al.23) and synthetic ACC (Radha et al. 7 supplement). The FSY and FSW HACC spectra lack the distinctive ν4 band for crystalline CaCO3, i.e., 712 cm−1 for calcite and 744 cm−1 for vaterite. Instead, the HACC spectra display low-intensity bands at 727 and 693 cm−1 that sit on a broad band centered near 600 cm−1. The IR spectrum shows the broad band between 2750-3800 cm−1 from absorbed and structural water, on which are superimposed weak absorption bands for organic material (inset Fig. S4). In comparison to the amorphous HACC, aged samples collected in the desert show FTIR spectra with characteristic absorption bands for crystalline CaCO3 (Figs. S4, S5).
The moisture stability of the FSY and FSW samples was investigated by placing the XRD slide with the sample used for powder XRD into a sealed container over water at 50°C for 24 hrs. The sample became damp over the 100% RH and a new powder XRD pattern was acquired. The FSY ACC diffraction pattern was unchanged, even after several 24 hr sessions of the 100% RH treatment. However, the FSW ACC showed the appearance of calcite reflections after one 24 hr session of 100% RH. These reflections became more intense with each 24 hr sessions of 100% RH, though no reflections for vaterite were noted (Fig. S6). In contrast, FSW samples collected after weeks to months of natural desert weathering are dominated by vaterite with minor calcite, to those dominated by calcite with minor vaterite: one peridium is composed of monohydrocalcite with minor amounts of calcite (Fig. S7).
Thermal response. The TG curves for the ACC samples studied under a He atmosphere show four distinct mass-loss steps, M1 to M4 (Figs. 5, S8, S9). The total mass losses for the FSW and FSY samples heated to 1000°C were typically between 52 and 57%. The DSC curves show an endothermic peak at ~100°C, corresponding to dehydration of the HACC and loss of loosely bound water (Fig. 5). The TG loss below 200°C is between 10.5 and 10.9% for the FSW samples (samples FSW21 and FSW18) and 11.2% for FSY. The EGA ion curves show that the mass loss below 200°C is dominated by the evolution of H2O with minor CO2 (Fig. 5, S10, S11). In region M2, the mass loss is dominated by H2O (3.8%), CO2 (Fig. S11) and organic molecules (Fig. S10d): the intense m/z=30 signal matches that of formaldehyde (see caption to Fig. S10 for additional ion peak assignments). During the M3 mass-loss step (between 396 and 584°C), organic matter is pyrolytically degraded (Fig. S12), with the evolution of a range of organic-bearing species, with minor H2O and CO2 (Fig. S10). Above 584°C, calcium carbonate decomposes, and the ion signals are dominated by m/z=44 corresponding to CO2 (Figs. 5, S10).
Quantity of water and organic matter. The FSY and FSW HACC shows TG mass losses up to 200°C of ~11 wt%, the majority of which is H2O. For example, of the 11.2% mass loss for FSY below 200°C (Fig. S11), 10.9±0.1% is from H2O, and the rest is CO2 (see Supplementary section - Water Calculation from TG-DSC-MSEGA measurements). A further 3.8% H2O is released in region M2 (Fig. S11), though only 3.0% up to the crystallization temperature onset at 322°C (see discussion below). However, considering the composition of the FSY HACC as inorganic Ca-C-O-O-O-H-H-O (equivalent to monohydrocalcite: CaCO3.H2O) + remaining C-H-N-O, then 13.72 wt% water loss (corresponding to 1 mole of water) is expected (Tables S3, S4). The lower-than-expected amount of water released below 200°C for FSY, i.e., 1CaCO3:nH2O ratio with n<1, may indicate significant molecular H2O retained above 200°C, but released before and during crystallization of the ACC starting near 322°C. Such a scenario is borne out by the TG data which shows a total H2O release up to 322°C of 13.9%. While the low-temperature water-loss peak has a maximum just above 100°C, which then drops precipitously with a plateau near 200°C, H2O continues to be released with increasing temperature and peaks near 320°C, with little H2O detected above 400°C (Figs. S10a, S11). These data suggest that the water released between 200 and 320° C has several sources including molecular H2O and that bound to the organic compounds.
The FTIR spectra show weak absorption bands between 2800 and 3000 cm−1 attributed to organic matter associated with the HACC (Fig. S4). The breakdown of this organic matter is also detected by EGA during heating of the HACC (Fig. S10). Samples heated in air have a higher mass loss than those heated under an inert atmosphere (Fig. S13). For example, FSY shows higher mass losses when heated in air between 200 and 575°C. In this temperature range, 19% of the mass is lost in the case of samples measured in air, and 15.1% when heated under He. This ~4% difference sets a minimum in the FSY sample on the organic content as some is pyrolytically degraded under He. The quantity of organic matter can also be estimated from the compositional data (Tables 1, S3 to S6). Assuming an inorganic formula for FSY as (Ca,Mn)1CO3.H2O, then the remainder is assumed to be organic and has the composition C0.60O0.09H1.41N0.14, which is 9.13% of the original HACC mass (Tables S3, S4). Similarly, the composition for FSW21 (Table 1) suggests it contains 9.83% organic matter (Tables S5, S6). Thus, the TG data is consistent with the F. septica HACC containing between approximately 4 and 10% organic matter.
Despite the elemental and compositional similarities between the F. septica HACC and monohydrocalcite (Table 1), their thermal behaviors differ significantly. Monohydrocalcite typically dehydrates by 226°C, with mass loss of 15.25% H2O, corresponding to CaCO3·H2O -> CaCO3 + H2O, and 37.26 wt% mass loss above 530°C for CaCO3 -> CaO + CO2 44,45. Monohydrocalcite shows minimal mass loss below ~180°C, and between ~200 and 500°C. In contrast, the slime mold ACC shows significant mass loss below 180°C, and also between 200 and 500°C. The slime mold thermal data are consistent with the different H environments, similar to that detected in ACC by Nuclear Magnetic Resonance spectroscopy 16, viz., fluid-like H2O, rigid structural H2O, restrictedly mobile H2O, and hydroxyl. Our mass loss up to 200°C is dominated by the loss of fluid-like H2O.
Laboratory crystallization of the DACC. The powder XRD data show onset of crystallization of the DACC between 300 and 350°C (Fig. S14). Similarly, SAED patterns and FTIR spectra of the HACC heated at 208°C show an amorphous material, but at 362°C the patterns and spectra show partial crystallization, and at 500°C the spheres are calcite (Fig. S15 to S17). At 362°C, the BFTEM images show that the newly formed calcite is ~40 nm, which is similar in size to the electron-dense aggregates imaged by BFTEM in the nanospheres (Fig. 2c, S3). By 500°C, the 40-nm nano-objects have organized into micron-sized grains and form calcite (Fig. S15). The spherical morphology is preserved during the transition (Fig. S15). Individual spheres show sharp extinction when rotated between the crossed polars, indicating that each is a single crystal and preserves the original spherical morphology.
The DSC profiles for HACC samples examined under He show peaks for exothermic reactions between 300 and 480°C (Fig. 5). The FSY sample shows prominent maxima at 327 and 401°C in the raw thermogram. Most of the FSW samples similarly reveal two well-resolved exothermic maxima near 338 and 414°C (Fig. S18). One sample (FSW21) lacks the 338°C maximum. The EGA data show a maximum in the evolution of a range of gases just prior to the first exotherm maximum for both the FSY (Fig. 6) and FSW (Fig. S19) precipitated HACC. Many of the gases associated with organic molecules show a two-peaked evolution with maxima at ~260 and ~320°C, whereas water and CO2 show a more gradual increase in signal intensity starting around 240°C, with a maximum near 320°C. The first exotherm between 327 and 338°C is within the crystallization range identified by powder XRD, and previously attributed to the crystallization of the dehydrated ACC 7,17,41,46, although this maximum can occur over a range of temperatures 16. Some biogenic ACCs show two exothermic peaks in the 330 to 370°C range 47. For example, the “Gastrolith ACC 2” DSC curve from 47 shows two exothermic peaks, of which one may be attributed to the breakdown of chitin 48. Between the first and second exotherm, the slime mold ACC continues to lose mass, typically between 1.3 and 3.5 wt%. However, additive free ACC does not typically show mass loss following the exothermic event near 340°C, indicating a solid-state transformation to calcite as no remaining water is available 7,17.
Our data suggests the breakdown of an organic framework to the DACC starting near 240°C, culminating with a maximum in several EGA signals just prior to the first DSC exotherm, with partial crystallization. The powder XRD data for the sample at 350°C still shows a significant amorphous contribution (Fig. S14). By 365°C, the powder XRD pattern shows intense reflections for calcite that sit on a less intense amorphous ACC background. Optical microscopy shows that at 365°C, approximately 20% of the spheres are isotropic. The DSC maximum near 400°C likely corresponds to the onset of crystallization of the remainder of the as yet amorphous part of the ACC, as optical microscopy and powder XRD of samples heated at 446°C show calcite only.
Multi-scale structure of the HACC. Our data for the F. septica HACC is consistent with the following structural spatial scales, viz., 1) the 500- to 1500-nm-sized sphere; 2) tens-of-nanometer-sized clumping within the spheres; 3) an ~2-nm-scale ordering as revealed by the powder XRD; and, 4) the short-range ordering that gives rise to the bulk powder XRD pattern. Some of the structural characteristics of the HACC may be explained by the formation mechanisms of the individual spheres. Electron microscopy shows the aggregation and excretion of calcium during sporogenesis in the slime mold Physarella oblonga 27, which is in the same family as F. septica, the Physaraceae 49. As the slime mold transforms from the mobile plasmodium to the sessile fruiting body there is a massive elimination of the protoplasmic Ca. During sporogenesis, electron microscopy reveals the formation and aggregation of tens-of-nanometers–sized Ca-rich, electron-dense, membrane-bound intracellular grains. These grains aggregate and assume their final spherical shape as they grow and are expelled and form the peridium 27. This membrane-bound aggregate model is consistent with our BFTEM of the F. septica spheres, which internally show tens-of-nanometer-scale electron-dense clumping (Figs. 2c, S3). However, the aggregates themselves are amorphous as the powder XRD profiles do not show reflections indicative of crystalline ordering at the tens-of-nanometer scale (Fig. S21).
The origin of the ~1.9 nm powder XRD maximum is obscure. This maximum becomes indistinct, or is absent, for samples heated to ~100°C, or stored over the aggressive drying agent P2O5. Thus, the 1.9 nm maximum may reflect ordering at this scale that is absent after loss of the weakly bound water. Ordered mesoporous material can give rise to low-angle maxima that reflect the pore-to-pore distance as well as the pore diameter 50. Thus, the ~1.9 nm maximum suggests an ordered mesoporous structure to the F. septica HACC that is readily lost on removal of the loosely bound water.
The powder XRD patterns for the room temperature and heated F. septica ACC lack discrete reflections for crystalline phases, and instead present broad maxima that reflects the short-range order within the spheres. These maxima represent the average interatomic distance scattering within the material and are indicative of short-range order. Rez et al. 8 show a strong match between the SAED patterns from synthetic and biogenic ACC and calculated patterns for random packing of ~1-nm-sized calcite crystals. Similarly, we compare the powder XRD patterns for the F. septica HACC with the simulated scattering profiles for ~1-nm-sized particles of anhydrous and hydrated CaCO3 polymorphs (Fig. 7, S20). Only the simulated pattern for monohydrocalcite shows four oscillations that match the 0.46, 0.2866, 0.204, and 0.119 nm maxima for the F. septica HACC powder XRD patterns. The simulated pattern for monohydrocalcite further shows a weak oscillation near 0.1467 nm which, if present in the experimental pattern, is obscured by the tail of the intense 0.288 nm peak. However, the 0.2866 nm peak in the experimental pattern is considerably more intense than the corresponding peak in the simulated pattern for monohydrocalcite. The intensity of this peak can be simulated by assuming an HACC structure with both monohydrocalcite and calcite like nano-structural ordering (Fig. 7). The main maximum for the HACC at 0.2866 nm is at a higher d-spacing than that predicted by the simulations. A similar situation was shown between the calculated patterns for calcite and biogenic calcite, which Rez et al. 8 attributed to the contraction of the nanocrystals relative to the bulk calcite. The simulations lend support to a structure composed of ~1-nm-sized diffracting domains, as the simulated patterns change dramatically just by doubling the particle size, with sharpening of the maxima and appearance of new peaks that are not present in the experimental patterns (Fig. S21).