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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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.

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 preparatio...

Discussion

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 b...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D-Printer	Stratasys	Objet 30 V3	Objet30 Pro V3.0 Desktop 3D-Printer
3D-Printer Material	Stratasys	OBJ-04066	Rigur RGD450 Model Material
3D-Printer Material	Stratasys	OBJ-04020	Sup 705 Support Material
Ammonium Chloride	Fisher Scientific	A661-500	Any other Ammonium Chloride should work, manufacturer should not matter
Brain Heart Infusion Broth	Millipore	53286	Any other Brain Heart Infusion Broth should work, manufacturer should not matter
Calcium Chloride Dihydrate	VWR	BDH9224	Any other Calcium chloride Dihydrate should work, manufacturer should not matter
Coarse Sand	Ward’s	470016-902	Special Sand-Gravel Mix and Stress Clay
Desktop Water Jet	Stratasys	OBJ-01400	Water jet system for post-processing of 3D prints
Epoxy	Gorilla Glue	4200102	GORILLA Epoxy Adhesive: Epoxy, 0.8 fl oz, Syringe, Clear, Thick Liquid
Fine Sand	Sandtastik	PLA25	Play Sand in Sparkling White
Gasket Material	McMaster-Carr	8525T65	Ethylene-propylene diene monomer (EPDM) 1/16” thickness
GrabCAD	Stratasys	GrabCAD	3D printer software
Magnets	K&J Magnetics	D64-N52	Neodymium Magnet Grade N52
SolidWorks 2021	Dassault Systèmes	SolidWorks 2021	CAD software
Sporosarcina pasteurii			Strain: ATCC 11859 / DSM 33
Vacuum Filtration cup 0.45µm	VWR	10040-450
Wire Mesh 1.5” Diameter Discs	McMaster-Carr	2812T43	Steel Wire Mesh Material