The single cultures and bacterial mixtures grown in YA broth overnight were harvested by centrifugation at 6000 × g for 5 min, washed twice, and resuspended in a 0.9% sodium chloride solution at a final OD600 of 1.0 (equivalent to 1 × 108 cells/mL). Sterile silica sand (200 g, 0.45–0.7 mm, Joomoonjin Sand Co. Ltd., Korea) was mixed with 10 mL urea (40 g/L) and calcium chloride dihydrate solution (25 g/L). The sand slurry (40 g) was packed into a 25 mL plastic column (Corning Co. Ltd., Corning, NY, USA). After being dried for 48 h at 50 ◦C, the columns were run once by gravity with 10 mL of cell suspension. The columns were stored for 48 h to allow calcite crystal growth. Then, 2 mL of crystal violet (CV) was pipetted onto the packed sand column. The degree of impermeability was determined by measuring the migration distance of CV.