Sandy soil reinforcement using carbonic anhydrase-producing bacterium with crude soybean urease system: Crystallinity optimization and extracellular polymeric substance function

Calcium carbonate crystals derived from enzyme-induced carbonate precipitation (EICP) typically display low crystallinity and inconsistent morphologies, which impede their binding efficacy in sandy soil stabilization. To overcome this challenge, we have developed a mineralization system that incorporates a carbonic anhydrase-producing bacterium (CPB) with crude soybean urease (CSUS) to enhance the reinforcement efficacy. We conducted comparative experiments in both liquid-phase and sand mineralization, using EICP as a benchmark. The reinforcement mechanism was elucidated by correlating the macroscopic mechanical properties of sand columns with the microstructural characteristics of the mineralized products. The results indicate that CPB significantly increases the quantity of calcium carbonate and its cementation quality at the contacts between sand particles, consequently enhancing the strength and rigidity of bio-cemented sand across various cementation levels. In samples with low cementation (0.5 M solution, four cycles), unconfined compressive strength increased by 229%, achieving 102 kPa. In contrast, with high cementation (1.5 M solution, eight cycles), strength improved by 20% to 3.5 MPa. Microscopic analyses demonstrate that CPB tightly associates with both sand particles and calcium carbonate through hydrogen bonds formed with amide groups in extracellular polymeric substances. The β-sheet conformation of these amide groups favors interactions with calcium ions, facilitating targeted deposition of calcium carbonate. CPB catalyzes the CO 2 hydration reaction, enhancing supersaturation in the mineralization system and supporting solute movement for crystal growth and refinement. This process fosters the development of calcite crystals with elevated crystallinity. Together, these multifaceted reinforcement mechanisms enable the CPB-CSUS system to substantially improve the mechanical performance and efficiency of reinforcement in bio-cemented sand.