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 ....

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 al....

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