Sporosarcina pasteurii can form nanoscale crystals on cell surface

Using a semi-solid 0.5% agar column, we study the phenomenon of microbially induced mineral (calcium carbonate) by the bacteria Sporosarcina pasteurii. Our platform allows for in-situ visualization of the phenomena, and we found clear evidence of bacterial cell surface facilitating formation of nanoscale crystals. Moreover, in the bulk agar we found the presence of microspheres, which seem to arise from an aggregation of nanoscale crystals with needle like morphology. Extensive chemical characterization confirmed that the crystals to be calcium carbonate, and two different polymorphs (calcite and vaterite) were identified.


Introduction
Biomineralization refers to the process of mineral precipitation due to chemical alteration of the environment induced by the microbial activity [1][2][3][4] . For unicellular organisms such as bacteria 4 , the biomineralization process can be either extracellular 5 or intracellular 6 . Microbially induced calcite precipitation (MICP) is an excellent example of an extracellular mineral deposition.
Several microbial species take part in MICP by means of various mechanisms such as photosynthesis 7,8 , urea hydrolysis 1,5 , sulfate reduction 9,10 , anaerobic sulfide oxidation 11 , biofilm and extracellular polymeric substances 12,13 . There has been significant interest in microorganisms that can produce urease (urea amidohydrolase; EC 3.5.1.5) and hence are able to hydrolyze urea to induce calcite precipitation 14 . Sporosarcina pasteurii (formerly Bacillus pasteurii) is a non-pathogenic, endospore producing soil bacteria that also produces urease and can tolerate highly basic environments. S. pasteurii has attracted significant attention from researchers for its unique feature of calcium carbonate precipitation, which can be easily controlled 10,[15][16][17][18][19] . S. pasteurii is being investigated for the possibility of its utilization towards a multitude of applications including underground storage of carbon, healing masonry structures of archaeological importance and long-term sealing of geologic cracks in large-scale structures 3

Results and Discussion:
Agar Column: To understand growth of S. pasteurii is a porous environment, an agar column with stab culture was observed for a period of one week. Figure 1(a) shows the original culture tubes containg growth spans after day-1 and day-7. Extensive calcite deposition can be prominently seen in Figure 1(b) after 7 days of incubation. The agar media acted as soft, porous and transperant nutrient riched environment to monitor the bacterial motility and calcite precipitation. Figure 1c depicts a scanning electron microscopy image of the agar column, where porous structures can clearly be seen. S. pasteurii bacterium possesses flagellum (see Figure   S1b) which likely allows it to navigate the porous structure of the agar column. The agar column was sectioned at a depth of 1.25 cm after 4 days, and the bacterial motion observed through optical microscopy. Figure

Discussion
In this work, we develop a simple, yet effective experimental platform for in-situ imaging of the MICP process by Sporosarcina pasteurii. Agar (0.5%) was used to create a porous column, which was inncoluated with a stab culture. As S. pasteurii cells migrated down the agar column they left conspicuous trail of crystals. Samples of the agar column at different locations were taken and subjected to microscopy and we found that the crystal train consisted of calcite microspheres, which on closer inspection were found to be an aggregate of needle-like nanoscale crystals. Moreover, cells whose surface contained calcite nanocrystals were also observed confirming the hypothesis that cell surface plays a role in nucleation.  35 . Nutrient Broth (NB) was prepared, which contained 5 g peptone; 3 g beef extract and 2 g sodium chloride per 1L of distilled water. The pH of this medium was adjusted to 7.0 using HCl and NaOH. Nutrient agar medium was prepared by utilizing the same ingredients as NB with an additional supplement of 1.5% bacteriological agar. water.

Materials and Methods
Preparation of semisolid-agar column: Semisolid-agar columns were prepared by following Bang's urea-CaCl2 liquid media with modification 1 . The modified media contain peptone 0.5 %; beef extract 0.3 %, sodium chloride 0.2 % and CaCl2, 0.28 %. The pH of the medium was adjusted to 7.0 and then 0.5 % agar was added prior to autoclaving. Urea (2 %) was added separately after autoclaving when media temperature cooled down to approximately 50-60° C.
To create the agar columns, 10 ml of liquid agar was poured into upright test tubes and allowed to cool inside a biosafety cabinet, which finally resulted in columns of approximately 5 cm in length. Subsequently, these agar columns were inoculated by stabbing the free surface of the agar column with pre-cultured S. pasteurii using a stabbing needle. Fresh liquid media were poured on to the agar column to prevent drying out from the agar surface. The bacteria inoculated columns along with a control were incubated at 30 °C for a maximum duration of 7 days. which was operated at 10 fps. Image analysis was performed using ImageJ.