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

Sporosarcina pasteurii is a ureolytic bacterium that breaks down urea into carbonate and ammonium. The carbonate combines with calcium to form calcium carbonate, creating a crystal lattice that anchors surrounding particles together to produce biocement. This is a convenient protocol for using 3D-printed molds to create biocement bricks suitable for compression testing.

Cement is a key building material used in many structures across the globe, from foundations for homes to historical monuments and roadways. It is a critical and abundant material worldwide. However, the traditional production of cement is a major contributor to man-made atmospheric CO₂, leading to greenhouse gas emissions and climate change. Microbially induced calcite precipitation (MICP) is a biological process in which Sporosarcina pasteurii or other bacteria produce a cement material that is as strong as traditional cement, but biocement is carbon-neutral. This MICP method of producing biocement is a promising technology and is currently under active investigation by many companies, countries, and research groups. The protocol presented here employs custom-designed, reusable, 3D-printed molds for flow-through MICP treatment of soil or sand, producing cylindrical bricks that meet standard specifications for unconfined compression tests. The individual, free-standing, reservoir-topped molds allow convenient parallel testing of multiple variables and replicates. This protocol outlines the S. pasteurii MICP reaction and the creation, assembly, and use of the 3D-printed molds to generate biocement cylindrical bricks.

Concrete is the main building material for construction projects around the world^1,2. One study found that cement is the second most consumed material in the world, behind only water³. Nearly 4.1 billion tons of cement are produced each year^4,5. Traditional production, processing, and application of cement results in nearly 8% of the global CO₂ emissions annually⁶. Due to the high demand and yet damaging effects of traditional cement production, a novel carbon-neutral method for cementation is a top priority for global sustainability goals^7,8,9,10.

Biocementation is the process of using microorganisms to produce a cement, adhesive, or substance that can be used to create a solid surface or structure^1,11. The most well-defined biocementation process involves using ureolytic bacteria to precipitate calcium carbonate, linking particles together into a hardened cement material^12,13.

When considering an eco-friendly alternative to traditional cement, the alternative must also meet the strength expectations for cement. The unconfined compression test is an analytical measurement used to determine the shear strength of a rock, building material, or soil sample¹⁴. For effective shear testing, the sample must be prepared according to industry standards, which include a 1:2 diameter-to-height ratio and a cylindrical shape¹⁵. A custom-designed 3D-printed mold was created to meet these standards and increase efficiency in executing an MICP protocol. These custom-designed molds allow for the flow-through application and drainage of sequential MICP treatments. Bacterial culture and cementation solution can easily be applied to the top reservoir, which then runs through the mold and passes through a mesh-lined opening on the base of the mold. The molds are designed to rest on top of a beaker or other waste collection container. The mold is split in half vertically to allow for easy unmolding of the cemented brick. It is held together by eight magnets affixed to the frame of the mold and sealed with epoxy to prevent damage to the magnets from exposure to the MICP solutions. The two halves also contain an inset groove to place a rubber gasket, which helps seal the mold and prevent leaking. On the inside of the cylindrical mold is a groove to indicate the fill level for sand/soil to produce a brick 3 inches in height; the space above that groove is intended to be used as a reservoir for the application of treatment solutions. A piece of wire mesh placed over the bottom opening on the inside of the mold, when constructed, prevents the sand or soil from falling out through the bottom of the mold. Additionally, a piece of wire mesh is placed on the top of the sand or soil to assist in evenly distributing the applied solutions and ensure the brick that is formed has an even top without any sharp ridges, which could affect the unconfined compression test results.

The molds were designed using computer-aided design (CAD) software, and an STL file (Supplementary File 1 and Supplementary File 2) was generated from the CAD file (Supplementary File 3 and Supplementary File 4). This STL file was uploaded into the 3D printer program and subsequently printed. After the molds were printed, a water jet system was used to remove the support material generated from the 3D printer, leaving the final 3D-printed structure. The file for printing a tamping device to aid in compacting the sand/soil in the mold and creating a level top surface has also been included.

The details of the reagents, equipment, and software used are listed in the Table of Materials.

1. Preparation of solutions and media

1. Brain-Heart Infusion (BHI) - urea medium (1 L)
   1. Weigh 37 g of BHI powder using a balance and add to a 1 L flask or beaker.
   2. Weigh 20 g of urea using a balance and add to the same 1 L flask or beaker containing BHI powder.
      CAUTION: Do not autoclave or add bleach to any materials containing urea. Urea will break down into ammonia, which can be harmful as a volatilized gas and can react with bleach to form toxic mustard gas. Dispose of all waste as hazardous waste per the institution's safety protocols.
   3. Fill the 1 L flask or beaker containing BHI powder and urea with 1 L of H₂O.
   4. Mix and filter sterilize the medium with a 0.45 µM filter into an autoclaved flask or beaker.
2. Cementation solution (1 L)
   1. Weigh 20 g of urea using a balance and add to a 1 L flask or beaker.
   2. Weigh 10 g of NH₄Cl (ammonium chloride) using a balance and add to the same 1 L flask or beaker containing urea.
      CAUTION: Do not autoclave or add bleach to any materials containing ammonium chloride. Ammonium chloride will form an equilibrium with ammonia gas, which can be harmful as a volatilized gas and can react with bleach to form toxic mustard gas. Dispose of all waste as hazardous waste per your institution's safety protocols.
   3. Weigh 49 g of CaCl₄.2H₂O (calcium chloride) using a balance and add to the same 1 L flask or beaker containing urea and ammonium chloride.
   4. Fill the 1 L flask or beaker containing urea, ammonium chloride, and calcium chloride with 1 L of H₂O.
      NOTE: This solution is not sterilized; prepare fresh and use within 48 h.
3. Brick printing and preparation (performed several days in advance of MICP treatment)
   1. Load the STL file for the brick mold (Supplementary File 1) and the tamping device (Supplementary File 2) to the appropriate program for the 3D printer.
      NOTE: The specific program used may be different using a different 3D printer. Use the appropriate program for the printer you are using.
   2. Print the molds and tamping devices (Figure 1).
   3. Process the molds according to the printer requirements.
   4. Place one magnet in each of the appropriate magnet slots in the mold, ensuring the charges are situated in such a way that the two halves of the mold attract and do not repel each other
   5. Once the magnets are appropriately placed, seal each magnet with epoxy.
   6. Select two 1.5-inch diameter circles of wire mesh and set aside.

2. Brick preparation (Day 0)

NOTE: The details for the preparation of one brick is provided here.

1. Filter sterilize 150 mL of BHI-urea medium. Autoclave a 250 mL flask.
2. Prepare 250 mL of cementation solution; do not place it in the autoclaved 250 mL flask.
3. Prepare S. pasteurii isolated streak culture on a Petri dish with BHI urea agar and incubate at 30° C for 24-48 h (S. pasteurii from frozen glycerol stock).
4. Starter culture of S. pasteurii (Day 1)
   1. Make a 1.6 mL starter culture by adding 1.6 mL of BHI-Urea medium to a culture tube.
   2. Inoculate the culture with 1 colony from the Day 0 streak plate.
   3. Grow the starter culture in a shaker (150 rpm) at 30 °C overnight.
5. Culture growth (Day 2)
   1. Inspect the starter culture to confirm growth (evident as increased turbidity).
   2. Add 40 mL of BHI-urea medium to the 250 mL autoclaved flask. Pour the 1.6 mL starter culture into the flask. Incubate and shake at 30 °C for 7 h.
   3. Add an additional 40 mL of BHI-urea medium to the flask. Place the flask in a shaker at 20 ° C overnight (~16 h).
6. Brick treatment with S. pasteurii (Day 3)
   1. Add an additional 40 mL of BHI-urea medium to the overnight culture flask and continue incubating the S. pasteurii at 20 ° C.
7. Prepare brick molds (Day 3) (see Figure 2).
   1. 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 connecting.
   2. Add a circle of fine wire mesh to the bottom of the cylindrical brick mold to stop sand from falling through the hole in the mold.
   3. Fill the mold with sand or other material up to the line on the inside of the mold and tamp firmly.
   4. Place another circle of wire mesh on the top of the sand to cover the entire top surface and tamp again.
   5. Place the mold on top of a waste container to catch the flow through.
8. Treatment procedure (Day 3)
   1. Pour 40 mL of S. pasteurii culture on top of the sand and allow it to soak in. Wait for 45 min.
   2. Pour 80 mL of cementation solution on top of the sand. Wait for 30 min.
   3. Pour 40 mL of S. pasteurii culture on top of the sand. Wait for 30 min.
   4. Pour 80 mL of cementation solution on top of the sand. Wait for 30 min.
   5. Pour 40 mL of S. pasteurii culture on top of the sand. Wait for 30 min.
   6. Pour 80 mL of cementation solution on top of the sand. Leave the brick alone for at least 48 h or until the sand appears dry.
9. Check the final product (Day 5).
   1. Open the molds carefully by splitting the mold in half and releasing the pressure from the magnets. Gently remove the brick from the mold.
      NOTE: If the sand seems wet, the mold will need to dry for another day or two before removing the brick from the mold (the drier the brick is, the easier it is to remove).
   2. Place the brick on a paper towel to continue to dry for 3 weeks before performing compression testing.
10. Cleaning of molds (Day 5)
    1. Once the brick is removed from the mold, separate the gaskets and wire mesh from each half of the mold.
    2. Soak the wire mesh in a solution of 70% ethanol for 24 h prior to rinsing with water. Slight scrubbing may be required to clean the mesh.
    3. Rinse the molds with 70% ethanol and scrub with a soft bristle brush, sponge, or other cleaning device at least 3 times; then clean with soap and water followed by air drying
    4. Rinse the gaskets with 70% ethanol and then clean them with soap and water, followed by air drying.

3. Compression testing (Day 25)

1. Analyze all bricks for strength using an unconfined compression test¹⁶.
   1. Ensure the circular ends of the brick are flat and even. If the ends are not even, use a file or other device to even out the surfaces.
      NOTE: The ends of the brick should be mostly flat if the wire mesh was applied correctly. It is critical that the ends of the brick are as even as possible to ensure an accurate measurement of strength.
2. Place a brick in a zippered or sealed plastic bag and position the brick in the plastic bag so the flat faces of the brick are not covered by a seam to achieve a smooth, flat coverage.
3. Place the brick on the lower loading plate. Place a flat and even loading plate on top of the brick.
4. Apply about 1 pound of pressure to the brick via the unconfined compression test machine.
5. Tare the digital readout.
6. Apply increasing load continuously according to machine specifications until complete structural failure of the brick is achieved.
7. Record the maximum weight-bearing load for each brick. Perform desired statistical analysis to evaluate the results.

Construction of the 3D-printed mold can be seen in Figure 1 and Figure 2. Positive results should be seen as a brick that retains its shape when removed from the mold and, following 3 weeks of drying, appears as a solid structure that can easily be handled with minimal material loss from touch. If the brick is not solid and there is crumbling or significant material loss from touch or movement, there may have been an error made in the media or culture preparation. Examples of positive and negative brick results can be seen in Figure 3.

As shown in Figure 4, the molds were used to simultaneously test two different substrates: coarse and fine sand. A total of four bricks using coarse sand and four using fine sand were made using the S. pasteurii protocol outlined here and subjected to unconfined compression testing. Previously documented results of unconfined compressive strength of biocemented soils using S. pasteurii indicate a range of 48-12,400 kPa depending on the soil or sand type and the urease activity of the S. pasteurii¹⁷. The average maximum load for the coarse sand bricks was 95.125 PSI (655 kPa), while the fine sand bricks withstood an average maximum load of 49.625 PSI (321.46 kPa). The ability to easily 3D print any number of molds as needed allowed for testing of all variables simultaneously, minimizing potential variations.

Figure 1: Brick mold. This figure illustrates the 3D printing map for the 3D-printed molds. Each half of the mold is printed separately. After the mold processing, magnets are placed in the designated eight spots and sealed with epoxy. The inner surface of the molds contains two recessed areas where the two halves connect. Rubber gasket material is cut to match these recessed areas to ensure a watertight seal for the mold. Please click here to view a larger version of this figure.

Figure 2: Mold construction, treatment, and demolding. This figure outlines the step-by-step process for assembling the molds and creating brick samples: Step 1: The mold is assembled by cutting the gasket material according to the file specifications, with each of the 16 magnets inserted into the designated holes and sealed with epoxy. Step 2: Gaskets are placed into the appropriate indentations in the mold. Step 3: The two halves of the mold are connected. Step 4: (a) A circular piece of wire mesh is inserted through the top of the mold to cover the bottom hole, preventing sand from falling through; (b) Sand or soil is added to the mold up to the fill line marked on the inside of the mold; (c) A second circular piece of wire mesh is placed on top of the sand or soil; (d) A tamping device is used to press firmly down on the top layer of wire mesh, ensuring a flat and even top layer for the brick. Step 5: The mold with the sand is positioned on top of a beaker or another container to collect the flow-through solution. Step 6: Treatments are applied following the protocol. Step 7: After the drying period, the mold is placed on its side, and the top half of the mold is carefully separated from the bottom half. If necessary, the brick is allowed to continue drying in the bottom half of the mold until it becomes solid enough to be lifted out in one piece. Please click here to view a larger version of this figure.

Figure 3: Expected results following the brick protocol. (A) shows the expected positive result, characterized by clear edges and a solid cylindrical structure. (B) shows the expected negative result, characterized by crumbling and a lack of structural stability. Please click here to view a larger version of this figure.

Figure 4: Compression testing. This figure presents the results of unconfined compression testing for eight bricks produced simultaneously using the 3D-printed molds. The coarse sand resulted in an average strength of 95.125 PSI, while the fine sand averaged 49.625 PSI. Error bars indicate the standard deviation. A Student's t-test was performed to calculate the p-value for statistical analysis. Bricks made with the coarse substrate were significantly stronger than those made with the fine particle-size substrate (p-value < 0.005). All bricks were treated with solutions from the same batch and dried under identical conditions to minimize experimental inconsistencies. Please click here to view a larger version of this figure.

Supplementary File 1: STL file for mold. This file contains the 3D printing STL file for the mold design. Please click here to download this File.

Supplementary File 2: STL file for tamping device. This file includes the 3D printing STL file for the tamping device. Please click here to download this File.

Supplementary File 3: Mold CAD file. This file provides the CAD file for the mold design. Please click here to download this File.

Supplementary File 4: Tamping device CAD file. This file contains the CAD file for the tamping device design. Please click here to download this File.

Critical steps
This biocementation protocol utilizes S. pasteurii MICP to produce biocemented cylindrical bricks that are suitable for unconfined compression testing. One of the most critical factors for unconfined compression testing is the shape and structure of the sample. Ensure that the top and bottom of the cylinder product are flat and the height of the brick is as close to 3 inches as possible; going slightly over the 3-inch height mark is better than going under. There is a bit of height lost when treatments are being applied due to the settling of the sand/soil; thus, it is recommended to slightly overfill the mold prior to initial treatment. The circle of wire mesh placed on the top of the sand/soil prior to treatments helps distribute the applied solution and create a more level surface¹⁶. Cleaning the molds, mesh, and gaskets thoroughly is critical to minimize the cross-contamination risks of future bricks. It is also important to clean the mesh or use new mesh because it will become biocemented/clogged over time and can reduce the flow-through rate if not cleaned or replaced^13,17.

Modifications/troubleshooting

Molds
Many other printing devices and materials may be used to meet researcher needs. The CAD file can also be modified to meet different needs and produce larger, smaller, or alternate mold shapes. Additionally, any gasket material or magnets could be used; just ensure they meet the dimensions in the CAD file or modify the CAD file to meet different needs. The wire mesh can also be swapped for a different mesh or potentially filter paper; ensure the pores are small enough to stop the particulates from falling through. If particulates fall through the bottom opening, this is often caused by improper placement of the wire mesh and the presence of a gap between the mesh and the mold. Check the placement of the mesh. If there is significant leaking of the applied solutions from the sides of the molds, it is likely an issue with the gasket material. There could have been an issue in the cutting process, or the gaskets could have been misplaced. If adjustment of the placement does not correct the issue, new gaskets may need to be cut. If cross-contamination is observed in the molds, soaking the molds, mesh, or gaskets in 70% ethanol solutions may be necessary, or replacing them with new molds, mesh, or gaskets^14,15.

MICP
The MICP application process can be modified to meet different needs, i.e., changing the flasks/beakers/ etc. The culturing process does not require the plate method described; liquid culture from a glycerol stock or any other culturing method may be applied here¹². Treatments can be applied to the soil sample using automatic pipettors or pouring from a graduated cylinder or any other means that allow for control of the volume. Sometimes, the bacterial cultures may not grow appropriately; this can be noted by the lack of turbidity following incubation. If this happens, restart the culturing process with a new colony or starter culture. A quantification step is advised, which is measuring OD600 or colony counts, to control and document the concentration of bacteria being applied to each brick¹⁶.

Limitations
This is a long process that takes several days and requires preparation prior to starting. There are no opportunities to pause the experiment once the Day 1 protocol has begun.

Significance
This protocol outlines a method of producing cylindrical biocement bricks suitable for unconfined compressive strength testing, providing a means to be able to test biocementation techniques for geotechnical applications^13,17.

Future applications
The importance of this protocol lies in its efficiency in optimizing biocementation protocols while simultaneously testing multiple variables in the process. The reusable molds allow the formation of cylindrical bricks of the specific dimensions used for unconfined compression testing, and the reservoirs at the top of the molds allow the MICP solutions to be applied in bulk instead of slowly applying solutions little by little while waiting for them to move through the material in the mold. Any number of individual molds can be printed and used in parallel, allowing easy comparison of different variables, such as changes in the chemical composition of cementation solution or the use of different microorganisms. Because the molds are designed to sit on top of a container for the collection of flow-through waste, the flow-through can be measured and assessed for bacterial count, pH, ion content, or any other test variable. Some studies, such as the evaluation of the MICP abilities of Escherichia coli genetically engineered to express the urease enzyme, have measured precipitation kinetics by measuring calcium depletion, with an emphasis on the direct comparison of different strains of bacteria or different plasmid constructs; this protocol is ideal for this type of evaluation or optimization research¹⁸.

The authors declare no conflict of interest. This manuscript has been approved for public release. PA number: USAFA-DF-2024-777. The views expressed in this paper are those of the authors and do not necessarily represent the official position or policy of the U.S. Government, the Department of Defense, or the Department of the Air Force.

This material is based on research sponsored by the United States Air Force Academy and Air Force Research Lab under agreement number FA7000-24-2-0005 (MG). The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes, notwithstanding any copyright notation thereon.