Real-time monitoring of calcification process by Sporosarcina pasteurii biofilm

Sporosarcina pasteurii is known to produce calcite or biocement in the presence of urea and Ca2+. Herein, we report the use of novel ultramicrosensors such as pH, Ca2+, and redox sensors, along with a scanning electrochemical microscope (SECM), to monitor a real-time, bacteria-mediated urea hydrolysis process and subsequent changes in morphology due to CaCO3 precipitation. We report that the surface pH of a live biofilm changed rapidly from 7.4 to 9.2 within 2 min, whereas similar fast depletion (10 min) of Ca2+ was observed from 85 mM to 10 mM in the presence of a high urea (10 g L−1) brine solution at 23 °C. Both the pH and the Ca2+ concentration profiles were extended up to 600 μm from the biofilm surface, whereas the bulk chemical composition of the brine solution remained constant over the entire 4 h of SECM experiments. In addition, we observed a change in biofilm surface morphology and an increase in overall biofilm height of 50 μm after 4 h of precipitation. Electron microscopy confirmed the changes in surface morphology and formation of CaCO3 crystals. Development of the Ca2+ profile took 10 min, whereas that of the pH profile took 2 min. This finding indicates that the initial urea hydrolysis process is fast and limited by urease or number of bacteria, whereas later CaCO3 formation and growth of crystals is a slow chemical process. The ultramicrosensors and approaches employed here are capable of accurately characterizing bioremediation on temporal and spatial scales pertinent to the microbial communities and the processes they mediate.