This study demonstrates the process of microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii. The method involves enriching bacterial cultures with specific nutrients to facilitate urease production, leading to the formation of calcium carbonate crystals.
Begin with a liquid culture of Sporosarcina pasteurii in a nutrient-rich medium.
Take an enrichment stock solution containing urea, calcium chloride, ammonium chloride, and sodium bicarbonate.
Add a portion of the enrichment medium to the bacterial culture tube.
Vortex the tubes to mix the bacterial cells and nutrients uniformly.
Incubate the enriched cultures in a non-shaking incubator.
Sporosarcina pasteurii, a Gram-positive soil bacterium, produces the enzyme urease in the presence of urea.
Urease breaks down urea into ammonium and carbonate ions.
Ammonium chloride and sodium bicarbonate help maintain an alkaline pH that supports mineral precipitation.
Carbonate ions react with calcium ions from calcium chloride to form insoluble calcium carbonate.
This precipitate begins to form around the bacterial cells and crystallizes into calcite, a stable crystal form.
These crystals settle as white deposits at the bottom of the tube, demonstrating microbiologically induced calcite precipitation, or MICP.
Transfer nine milliliters of the prepared liquid culture into several sterilized ten milliliter centrifuge tubes. Prepare 100 milliliters of the external enrichment stock solution by using an analytical balance to carefully measure out two grams per liter of urea, one gram per liter of ammonium chloride, 212 milligrams per liter of sodium bicarbonate, and 280 milligrams per liter of calcium chloride.
Into a beaker with fresh medium, combine all the ingredients except the urea, and autoclave. After autoclaving, mix the urea with one milliliter of fresh medium and pass the solution through a syringe fitted with a 0.2 micrometer filter before adding to the enrichment medium. Add one milliliter of the enrichment medium with additives to the sterilized centrifuge tubes containing nine milliliters of the prepared culture.
Vortex each tube and incubate in a non-shaking incubator at 30 degrees Celsius. Monitor all the units regularly for initiation of precipitation.
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