Not Always Calcite: How Three Sporosarcina Strains Produced Different Calcium Carbonate Phases (Clarà Saracho et al., 2020)

The usual account of Microbially Induced Calcite Precipitation (MICP) puts the mineral in its name: bacteria split urea and calcite binds the grains. A 2020 open-access paper in Scientific Reports by Alexandra Clarà Saracho, Stuart K. Haigh, Toshiro Hata, Kenichi Soga, Stefan Farsang, Simon A. T. Redfern and Ewa Marek tests that assumption. Three closely related ureolytic bacteria were given the same treatment, and they did not make the same mineral. One produced calcite, one a calcite–vaterite mixture, and one mostly amorphous calcium carbonate (ACC). The authors say their results suggest that polymorphism in biological carbonate systems is "far more common than previously anticipated", and they argue this matters for soil improvement. That suggestion rests on three Sporosarcina strains grown in test tubes.

Background: calcium carbonate has several forms

Calcium Carbonate (Calcite, CaCO₃) can crystallise as calcite, aragonite or vaterite. The paper ranks them from most soluble and least stable (vaterite) to least soluble and most stable (calcite). There are also hydrated, metastable forms, including ACC and a hemihydrate (CaCO₃·1/2H₂O). In the sequence ACC → vaterite → calcite, the unstable forms are usually treated as short-lived intermediates. The authors cite earlier work suggesting that organic molecules from bacteria can halt this sequence partway, which would leave vaterite or ACC in the finished material.

What was done

The three strains, all from the genus Sporosarcina, came from different habitats:

• S. pasteurii (ATCC 11859), from dry surface conditions. The paper calls it the most common soil bacterium in geotechnical work.
• S. aquimarina (ATCC BAA-723), from shallow sea.
• S. newyorkensis, a deep-sea isolate from offshore Japan obtained from pressure cores. The authors say its precipitation behaviour had not been studied before.

Each strain was grown at atmospheric pressure and 30 °C to an optical density (OD₆₀₀) of about 0.5. Each suspension was then mixed 1:2 with a cementation solution of 0.3 M urea and 0.3 M calcium chloride plus 3 g/L nutrient broth, in 14 mL test tubes. The precipitates stayed in solution for more than 30 days and were then dried. They were characterised by X-ray diffraction, Raman spectroscopy, thermogravimetric analysis with mass spectrometry of the released gases, and electron microscopy. Calcium concentration and pH were followed over time. A second set of experiments with S. pasteurii alone varied the bacterial density and the solution strength (0.02, 0.2 and 1 M). A third grew all three strains for four days at 1 atm or 3 MPa and at 4, 15 or 30 °C to see how growth and urease activity responded.

What the three strains produced

• S. newyorkensis: mainly calcite. Its carbonate decomposition step at 757 °C removed 38.1 wt%, which the authors read as near-pure CaCO₃.
• S. aquimarina: calcite with vaterite as a secondary phase. The precipitate held 8.0 wt% structural water. Raman spectra taken into a single crystal to a depth of 25 μm showed calcite at the surface and vaterite's double peak (1076 and 1088 cm⁻¹) inside. Electron microscope images of the loose precipitate showed spherulites 5–50 μm in diameter, with rod-shaped cells embedded in the growing spheres. Separately, an epoxy-cast cross-section revealed hollow cores, some still holding traces of vaterite spherulites. From the depth profile of one crystal and this one cross-section, the authors infer that the calcite grew from a metastable vaterite precursor and not from a crystalline seed. That is their interpretation, and the paper itself notes that calcite and vaterite Raman peaks overlap in this region.
• S. pasteurii: no calcite peaks at all. X-ray diffraction showed only a broad hump typical of ACC. The carbonate decomposition step accounted for only 8 wt% of mass loss, compared with 27 wt% for water loss and ACC crystallisation and 18 wt% attributed to breakdown of organic or other macromolecules.

How the authors explain it

Kinetics. The strains had different specific urease activities at the same starting density, and their solutions changed at different rates. In the paper's account, S. pasteurii removed calcium from solution fastest at first and reached the highest pH (about 7.8–8.3), which stabilised ACC. S. newyorkensis nucleated most slowly at a near-neutral pH (about 7–7.5). Calcium returned to solution within the first 24 hours and then precipitated again as calcite, which the authors read as a direct ACC-to-calcite route. S. aquimarina fell between the two (pH about 7.5–7.8) and followed an ACC → vaterite → calcite path. The paper describes S. pasteurii as the low-urease strain and S. newyorkensis as the high-urease one. It also says S. pasteurii hydrolysed more urea in the early period. The text does not reconcile these two statements, and the activity values themselves are in a supplementary table.

Water and amino acids. Gas analysis during heating released ammonia, which the authors take as indirect evidence that amino acids were trapped in the precipitates. Organic matter alone did not explain the vaterite, because the calcite-only S. newyorkensis precipitate also contained amino acids. The authors propose that vaterite survives only when structural water is present as well. In their model, the water is hydrogen-bonded to particular polar amino acids at crystal edges. They say that confirming this "requires direct amino acid analysis", which they did not carry out.

Dose and density. With S. pasteurii, the 0.02 M treatments gave ACC-like precipitates. The 0.2 M treatments gave vaterite and calcite with water content consistent with a hemihydrate. At 1 M, high bacterial density gave only calcite and diluted cultures gave vaterite. The authors conclude that calcite alone needed both a strong solution and many cells.

Pressure and temperature. Temperature affected growth more than pressure did. Raising pressure to 3 MPa reduced specific urease activity in all three strains. The highest activities came from different settings for each strain: S. newyorkensis at 4 °C after one day, S. aquimarina at 15 °C after one day, and S. pasteurii at 30 °C after four days.

Why it matters for bio-cement

In the authors' framing, geotechnical MICP has generally reported calcite as the only phase. They argue that different phases give different properties at the contacts between grains, and therefore different strengthening effects. Citing earlier numerical and experimental studies, the paper notes that heavier cementation and greater inter-particle friction both push cemented sand from ductile towards brittle behaviour, an unwanted response because brittle materials fail catastrophically. The authors also link this to Ureolysis-Driven Carbonate Precipitation's main drawback. Getting calcite alone from S. pasteurii required strong solutions and high cell densities, conditions the paper says would yield substantial amounts of ammonium, which it calls a hazardous by-product. In their view, a strain like S. newyorkensis, which made calcite at lower inputs, could reduce that by-product (see Rinsing Out the Ammonium: A 3.7-Metre Test of By-product Removal After Ureolytic Bio-Cementation (Lee et al., 2019) for what removing ammonium afterwards involves). They also see the pressure sensitivity of urease as relevant to any MICP use in the deep sea.

What the study does and does not establish

All the work was done in test tubes. No sand was cemented and no strength, stiffness or durability was measured. The strength discussion relies on earlier numerical and experimental literature, not on this study's data, so it remains a hypothesis about Bio-Cementation. The amino-acid and water mechanism rests on indirect thermal and spectroscopic evidence. The 0.02 M samples were too small for X-ray diffraction, and the authors inferred their phase from similar heating behaviour. The authors also say their three strains are not necessarily representative of carbonate-precipitating bacteria in general. They do not report how long the vaterite or ACC lasts after the 30-day period. The work was supported by an EPSRC doctoral training centre at the University of Cambridge and was carried out as part of the MH21 methane hydrate consortium's activity in Japan, and the authors declare no competing interests.