This work was supported by the National Science Foundation Grant No. 1531382 and MarTREC.

NOTE: All relevant material used in the following procedures are non-hazardous. Personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes) are still needed.
1. Preparation of bacteria solution
<ol>
	<li>Preparation of growth medium (NH4-YE medium) 
	NOTE: The components of growth media per liter of deionized water are: 20 g of yeast extract; 10 g of (NH4)2SO4; and 0.13 M Tris buffer (pH 9.0).
	<ol>
		...

<ol>
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T., Brown, D. G., Kavazanjian, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+behavior+of+sands+treated+by+microbially+induced+carbonate+precipitation.">Mechanical behavior of sands treated by microbially induced carbonate precipitation.</a> Journal of Geotechnical and Geoenvironmental Engineering. 142 (2), 04015066 (2015).</li><li>Lauchnor, E. G., Topp, D. M., Parker, A. E., Gerlach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+cell+kinetics+of+ureolysis+by+sporosarcina+pasteurii.">Whole cell kinetics of ureolysis by sporosarcina pasteurii.</a> Journal of Applied Microbiology. 118 (6), 1321-1332 (2015).</li><li>Nafisi, A., Montoya, B. 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The MICP technique by immersion was presented in this paper. Soil samples were immersed into the batch reactor to get fully penetrated by cementation media in the MICP process. In this method, a full contact flexible mold, a rigid full contact mold, and a cored brick mold were applied to prepare MICP-treated samples.
Different molds can be designed for different geometry requirements. The fibrous structure of geotextile increased the contact area between sand and cementation media, which effec...

As a biological ground improvement technology, microbially induced calcite precipitation (MICP) is capable of improving engineering properties of soil. It has been used to enhance the strength, stiffness, and permeability of soil. The MICP technique has gained much attention for soil improvement worldwide1,2,3,4. Carbonate precipitation naturally happens and can be induced by nonpathogenic organisms that are native to the soil environment5. The MICP biogeochemical reaction is driven by the existence of ureolytic bacteria, urea and a calcium-rich solution5,6. Sporosarcina pasteurii is a highly active urease enzyme that catalyzes the reaction network towards precipitation of calcite7,8. The urea hydrolysis process produces dissolved ammonium (NH4+) and inorganic carbonate (CO32-). The carbonate ions react with calcium ions to precipitate as calcium carbonate crystals. The urea hydrolysis reactions are shown here:
<img alt="Equation 1" src="/files/ftp_upload/60059/60059equ01.jpg" />
<img alt="Equation 2" src="/files/ftp_upload/60059/60059equ02.jpg" />
The precipitated CaCO3 can bond the sand particles together to improve the engineering properties of MICP-treated soil. The MICP technique has been applied in various applications, such as improvement of strength and stiffness of soil, repair of concrete, and environmental remediation9,10,11,12,13,14,15.
Zhao et al.16 developed an immersion method to prepare MICP-treated samples. A full contact flexible mold made of geotextile was used in this method. The precipitated CaCO3 distributed uniformly throughout their MICP-treated samples. Bu et al.17 developed a rigid full contact mold to prepare MICP-treated beam samples by an immersion method. The MICP-treated sample prepared by this method using a rigid full contact mold can form the suitable beam shape. The MICP-treated sample was divided into four and the CaCO3 contents were measured. The CaCO3 content ranged from 8.4 &#177; 1.5% to 9.4 &#177; 1.2% by weight, which indicated that the CaCO3 distributed uniformly in the MICP-treated samples by the immersion method. These MICP-treated samples also achieved better mechanical properties. These MICP-treated bio-specimens reached a 950 kPa flexure strength, which was similar to that of 20- 25% cement-treated samples (600- 1300 kPa). Li et al.10 added randomly distributed discrete fiber into the sandy soil and treated the soil by the MICP immersion method. They found that the shear strength, ductility, and failure strain of MICP-treated soil were enhanced obviously by adding appropriate fiber.
The immersion method for MICP has been continually improved10,16,17. This method can be used to prepare MICP-treated soil samples and MICP-treated prefabricated building materials, such as bricks and beams. Different geometry dimensions of sample preparation mold were developed. Fibers were added in the MICP-treated samples to enhance their properties. This detailed protocol was intended to document the immersion methods for MICP treatment.

<table>
	<tbody>
		<tr>
			<td>Ammonium Chloride, &#62;99%</td>
			<td>Bio-world</td>
			<td>40100196-3 (705033)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulfate</td>
			<td>Bio-world</td>
			<td>30635330-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate, &#62;99%</td>
			<td>Bio-world</td>
			<td>40300016-3 (705111)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Broth</td>
			<td>Bio-world</td>
			<td>30620056-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate, &#62;99%</td>
			<td>Bio-world</td>
			<td>41900068-3 (705727)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sporosarcina pasteurii</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 11859</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic fiber</td>
			<td>FIBERMESH</td>
			<td>Fibermesh 150e3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Base, Biotechnology Grade, &#62;99.7%</td>
			<td>Bio-world</td>
			<td>42020309-2 (730205)</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea, USP Grade, &#62;99%</td>
			<td>Bio-world</td>
			<td>42100008-2 (705986)</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Bio-world</td>
			<td>30620096-3 (760095)</td>
			<td></td>
		</tr>
	</tbody>
</table>

sandy soil improvement through microbially induced calcite precipitation (micp) by immersion

The goal of this article is to develop an immersion method to improve the microbially induced calcite precipitation (MICP) treated samples. A batch reactor was assembled to immerse soil samples into cementation media. The cementation media can freely diffuse into the soil samples in the batch reactor instead of cementation media being injected. A full contact flexible mold, a rigid full contact mold, and a cored brick mold were used to prepare different soil sample holders. Synthetic fibers and natural fibers were selected to reinforce the MICP-treated soil samples. The precipitated CaCO3 in different areas of the MICP-treated samples was measured. The CaCO3 distribution results demonstrated that the precipitated CaCO3 was distributed uniformly in the soil sample by the immersion method.

Figure 7 shows the distribution of precipitated CaCO3 throughout the MICP-treated sample. The MICP-treated sample was divided into three different areas. The CaCO3 content in each area was tested by the acid washing method. To dissolve precipitated carbonates, the dry MICP-treated samples were washed in a HCl solution (0.1 M), then rinsed, drained, and oven-dried for 48 hours. The difference value between the masses of samples before and after acid washing was considere...

Watch this Scientific Journal Video about Sandy Soil Improvement through Microbially Induced Calcite Precipitation MICP by Immersion at JoVE.com

Sandy Soil Improvement through Microbially Induced Calcite Precipitation MICP by Immersion

Here, microbially induced calcite precipitation (MICP) technology is presented to improve soil properties by immersion.

Sandy Soil Improvement through Microbially Induced Calcite Precipitation (MICP) by Immersion

The goal of this article is to develop an immersion method to improve the microbially induced calcite precipitation (MICP) treated samples. A batch ...

The immersion method demonstrated in this protocol help researcher remove unbroken MICP-treated soil sample for mechanical testing through different mold. This technique help MICP-treated soil sample get more uniform precipitated calcium carbonate. Those MICP-treated soil samples also get better mechanic properties through this method.The most important aspect of this technique is to ensure the activity of bacteria is good enough for MICP process to avoid time consuming and material wasting. Designing appropriate molds for different application is considered to be another important aspect for researchers who intend to select this technique for the first time. To begin, transfer 0.1 milliliter of bacterial suspension to a centrifuge tube filled with 10 milliliters of fresh growth medium.Mix well by inverting and then, loosen the lid of the tube in order to maintain aerobic conditions. Prepare six tubes of bacterial suspensions with growth medium and one control tube with 10 milliliters of fresh growth medium alone. Incubate all tubes in a shaker at 200 RPM and 30 degrees Celsius for 48 to 72 hours.If the growth medium becomes turbid after 48 hours, stop the incubation. Centrifuge the tubes with bacteria and growth medium at 4, 000 times g for 20 minutes. Remove the supernatant, replace with 25 milliliters of fresh growth medium, and mix well using a vortex machine.Then, to enhance the culture of bacteria, transfer 0.25 milliliters of the suspension from the tubes into new tubes with 25 milliliters of growth medium to inoculate. Incubate all tubes in a shaker at 200 RPM and 30 degrees Celsius for 48 hours. Centrifuge the tubes with bacteria and growth medium at 4, 000 times g for 20 minutes.Remove the supernatant, replace with fresh growth medium, and mix well using a vortex machine. Measure the optical density of the suspension at 600 nanometers on a spectrophotometer. Adjust the bacteria concentration using fresh growth medium to an OD600 of 0.6.Add approximately 140 grams of dry Ottawa sand with 99.7%cords into molds by the air pluviation method to reach a median dense condition with relative density in the range of 42 to 55%and sand dry density in the range of 1.58 to 1.64 grams per cubic centimeters. Place a cover on top of the samples and fix it by sewing. Pour the bacterial solution through the permeable geotextile cover into the samples.When flowing bacterial solution is observed on the bottom of soil samples, they are considered saturated. Next, place the samples on the shelf. Immerse the entire shelf into a batch reactor filled with cementation media.Turn on the air supply and adjust the air output to keep 100%air saturation. Wait for seven days of MICP reaction. Take out the samples from the reactor.Use a knife to cut the full-contact flexible mold. Then, wash the samples with water to remove the residual solution in the pore spaces. Place the samples in a 105-degree Celsius oven for 48 hours until their weights remain constant.For the synthetic fiber, mix 2.7 grams of the fibers and 900 grams of the dry sand in small increments by hand to obtain a uniform mixture with 0.3%fiber content. For the natural palm fiber, prepare four parts of sand with 190 grams each. Prepare three layers of fibers with approximately 1.8 grams each.Add the four parts of sand and three layers of fibers in rigid full-contact mold at intervals. Next, repeat the MICP treatment as previously. In this protocol, precipitated calcium carbonate was observed throughout the MICP-treated sample.The MICP-treated sample was divided into three different areas. The calcium carbonate content of the MICP-treated sample by the immersion method ranged from 9.0 to 9.5%indicating the precipitated calcium carbonate was distributed uniformly throughout the soil sample. The stress strain curves of bio-brick reinforced with three layers of palm fiber and unreinforced bio-brick showed the flexure strength of unreinforced bio-brick was 1, 150 kilopascals, while that of reinforced bio-brick was 980 kilopascals.The flexure strain was improved significantly by addition of the palm fiber. It is very important to ensure the activity of bacteria solution before MICP process. If not, it is uncertain to be successful in the experiment.Once the experiment fails, it should be a waste of time and materials. Following this procedure, different molds can be designed for different experimental purpose through MICP technique. The influence of MICP on different kinds of structure or different materials can be simulated by these molds.

sandy-soil-improvement-through-microbially-induced-calcite

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JoVE Journal

Engineering

9.2K visualizações.  Jackson State University. O método de imersão demonstrado neste protocolo ajuda o pesquisador a remover amostras de solo ininterruptas tratadas com MICP para testes mecânicos através de diferentes moldes. Esta técnica ajuda a amostra de solo tratada com MICP a obter carbonato de cálcio precipitado mais uniforme. Essas amostras de solo tratadas com MICP também recebem melhores propriedades mecânicas através deste método.O aspecto mais importante desta técnica é garantir que a atividade das bactérias...