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Method Article
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 world1,2. 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 year4,5. 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 goals7,8,9,10.
Biocementation is the process of using microorganisms to produce a cement, adhesive, or substance that can be used to create a solid surface or structure1,11. The most well-defined biocementation process involves using ureolytic bacteria to precipitate calcium carbonate, linking particles together into a hardened cement material12,13.
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
2. Brick preparation (Day 0)
NOTE: The details for the preparation of one brick is provided here.
3. Compression testing (Day 25)
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...
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...
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.
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 |
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