Production and Analysis of Sporosarcina pasteurii Biocement Bricks Using Custom 3D-Printed Molds for Unconfined Compression Tests

The research investigates microbially induced calcite precipitation using S.pasteurii to produce biocement bricks. It explores the efficacy of 3D printed molds in optimizing biocement production and strength testing. Currently this experiment requires a significant time commitment as it takes approximately five days start to finish, and there's no ideal time to pause the experiment once started.

Our protocol uses reusable 3D printed molds for flow through MICP treatment, enabling standardized cylindrical bricks for unconfined compression tests while allowing convenient parallel testing of multiple variables and replicates. To begin, filter sterilize 150 milliliters of BHI urea medium and autoclave a 250 milliliter flask. Prepare 250 milliliters of cementation solution.

Add 1.6 milliliters of BHI urea medium in a culture tube and inoculate the medium with one colony from the day zero streak plate. Grow the starter culture in a shaker at 150 revolutions per minute at 30 degrees Celsius overnight. The next day, inspect the starter culture for growth, which is confirmed by increased turbidity.

Now, add 40 milliliters of BHI urea medium to the 250 milliliter autoclaved flask. Pour the 1.6 milliliter starter culture into the flask and incubate it in a shaker at 30 degrees Celsius for seven hours. After incubation, add an additional 40 milliliters of BHI urea medium to the flask, and place the flask in a shaker at 20 degrees Celsius overnight, or for 16 hours.

Now, add an additional 40 milliliters of BHI urea medium to the overnight culture flask and continue incubating Sporosarcina pasteurii at 20 degrees Celsius. Place rubber gaskets in the appropriate spaces on the molds. Connect the two halves of the molds, ensuring the gaskets are sealed and all magnets are connected.

Add a circle of fine wire mesh to the bottom of the cylindrical brick mold to prevent sand from falling through the hole. Now, fill the mold with sand or other material up to the internal line, and tamp firmly. Then, place another circle of wire mesh on the top of the sand to cover the entire surface and tamp again.

Now, position the mold on top of a waste container to catch flow through. Pour 40 milliliters of the culture on top of the sand and allow it to soak in for 45 minutes. Then, pour 80 milliliters of cementation solution on top of the sand.

After 30 minutes, pour 40 milliliters of culture on top of the sand, and wait for another 30 minutes. Again, pour 80 milliliters of cementation solution on top of the sand and then pour 40 milliliters of culture on top after 30 minutes. After half an hour of incubation, pour 80 milliliters of cementation solution on top of the sand and leave the brick undisturbed for at least 48 hours or until the sand appears dry.

On day five, split the mold in half and release the pressure from the magnets to remove the brick from the mold. Then place the brick on a paper towel to continue drying for three weeks before performing compression testing. For the compression test, place the brick on the lower loading plate of the unconfined compression test machine and a flat loading plate on top of the brick.

Using the compression test machine, apply about one pound of pressure to the brick, then use the digital readout to reset the pressure measurement. Finally, apply increasing load continuously according to machine specifications until the brick reaches structural failure. Record the maximum weightbearing load for each brick.

The 3D printed mold successfully retained the shape of the brick, producing bricks that appeared solid after three weeks of drying with minimal material loss upon handling. Bricks that exhibited crumbling or significant material loss upon touch indicated an error in media or culture preparation. Molds were used to test coarse and fine sand substrates, producing four bricks for each type.

The average maximum load for coarse sand bricks was 95.125 pounds per square inch, while fine sand bricks withstood an average maximum load of 49.625 pounds per square inch. The ability to 3D print molds as needed allowed for simultaneous variable testing, reducing experimental variation.