Optimization of Flocculation-Based Seed Culture Solution Preparation and Storage for Microbially Induced Carbonate Precipitation

Microbially induced carbonate precipitation (MICP) is a promising biogeotechnical technology, but its large-scale application is constrained by the high cost and energy consumption of bacterial cultivation and harvesting. This paper presents a metal ion-induced flocculation strategy for the preparation and storage of high-density Sporosarcina pasteurii seed cultures. The flocculation performance and biological effects of Ca2+ and Fe3+ were systematically compared. Ca2+ achieved efficient flocculation through electrical double-layer compression while maintaining near-neutral pH, thereby preserving urease activity and bacterial viability. Even at an inoculation ratio of 1:10,000, the recovered bacteria exhibited rapid regrowth and high urease activity. In contrast, although Fe3+ induced rapid flocculation at low concentrations, the acidic conditions severely impaired bacterial viability and regrowth. Cryopreservation with glycerol at −20 °C maintained bacterial activity for at least 90 days and markedly outperformed storage at 4 °C. The recovered flocculated bacteria exhibited CaCO3 precipitation performance comparable to that of freshly cultured suspensions. This study provides a simple, economical, and scalable strategy for the preparation and long-term storage of MICP seed cultures, facilitating the practical implementation of large-scale biogeotechnical applications.