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>
		<li>Autoclave ingredients separately.</li>
		<li>Dissolve 20 g of yeast extract, and 10 g of (NH4)2SO4 in 1 L of deionized water containing 0.13 M Tris buffer.</li>
		<li>Mix the components together using magnetic stirrer post-sterilization.</li>
	</ol>
	</li>
	<li>Propagation procedure of Sporosarcina pasteurii 
	NOTE: Use 50 mL centrifuge tubes in this experiment.
	<ol>
		<li>Thaw the frozen bacteria in a vial.</li>
		<li>Open the vial.</li>
		<li>Transfer 0.1 mL of bacteria suspension to a centrifuge tube with 10 mL of fresh growth medium. Mix well by hand (inoculation rate is 1:100). Repeat 5 more bacterial suspensions with growth medium. Prepare a control tube only with 10 of fresh growth medium inside. 
		NOTE: The cryoprotectant used in the freeze/drying procedure may inhibit growth in the primary tube. The lids of tubes were tightened loosely in order to maintain the aerobic condition.</li>
		<li>Incubate all tubes in a shaker at 200 rpm at 30 &#176;C for 48 to 72 hours. Stop the incubation if the growth medium becomes turbid after 48 h. Otherwise, extend the incubation to the maximum of 72 h.</li>
		<li>Centrifuge the tubes with bacteria and growth medium at 4,000 x g for 20 min.</li>
		<li>Remove the supernatant, replace with 25 mL of fresh growth medium, and mix well using a vortex machine.</li>
		<li>Repeat steps 1.2.3-1.2.6 twice to fully stimulate the activity of bacteria.</li>
		<li>Use the suspension from the tubes in step 1.2.7 to inoculate more tubes with 25 mL of growth medium to enhance the culture of bacteria (inoculation rate is 1:100).</li>
		<li>Incubate all tubes in a shaker at 200 rpm at 30 &#176;C for 48 hours.</li>
		<li>Centrifuge the tubes with bacteria and growth medium at 4,000 x g for 20 min.</li>
		<li>Remove the supernatant, replace with fresh growth medium, and mix well using a vortex machine.</li>
		<li>Adjust the bacteria concentration using fresh growth medium before the MICP experiments. Calculate the bacteria concentration by optical density of the suspension at 600 nm, which was measured using a spectrophotometer. The OD600 in this experiment was 0.6.</li>
	</ol>
	</li>
</ol>
2. Preparation of cementation media
NOTE: Cementation media is used to provide chemicals to induce the calcite precipitation during the MICP treatment. The urea-Ca2+ molar ratio is 1:1. The chemical components of cementation media is shown in Table 1. The following procedure is for 20 L of cementation media with 0.5 M Ca.
<ol>
	<li>Prepare 20 L of water in a plastic box.</li>
	<li>Dissolve 200 g of NH4Cl, 60 g of nutrient broth, 42.4 g of NaHCO3, 600 g of urea, and 1470 g of CaCl2&#8729;2H2O in the 20 L of distilled water. Mix well using stirring rod.</li>
</ol>
3. Preparation of molds
<ol>
	<li>Preparation of full contact flexible mold (FCFM) 
	NOTE: The full contact flexible mold is made of geotextile. The geotextile has a grab tensile strength of 1,689 N, a trapezoidal tear strength of 667 N, an apparent opening size of 0.15 mm, a water flow rate of 34 mm/s, a thickness of 1.51 mm, and a unit mass of 200 g/m2. The size of mold can be varied to prepare different sample sizes (for example, unconfined compression test sample or direct shear test sample).
	<ol>
		<li>As the FCFM consists of an annular part, a bottom, and a cover, cut the geotextile into constituent parts of FCFM.</li>
		<li>Sew the three parts of FCFM together as shown in Figure 1.</li>
	</ol>
	</li>
	<li>Preparation of rigid full contact mold (RFCM) for bio-bricks 
	NOTE: The rigid full contact mold consists of a flexible layer and a rigid holder. The flexible layer is made of the same geotextile as the FCFM. The rigid holder is made of a polypropylene perforated sheet with 6.35 mm diameter staggered holes distributed on the polypropylene perforated sheet and the clearance distance between adjacent holes is 9.53 mm. One mold consist of three chambers and the size of each chamber is 177.8 mm in length, 76.2 mm in width and 38.1 mm in height. The size of RFCM can be varied to prepare different sample size. The holes in the rigid holder allow cementation media flow through the flexible layer freely.
	<ol>
		<li>Prepare the polypropylene perforated sheet for constituent pieces of the rigid holder.</li>
		<li>Assemble the pieces of rigid holder using plastic screws and nuts.</li>
		<li>Prepare the constituent parts of the geotextile flexible layer. The flexible layer consists of a bottom and a cover.</li>
		<li>Enclose the bottom of the flexible layer in the rigid holder.</li>
		<li>Once the sand is added into the mold, place the cover of flexible layer and fix by sewing on the top of the sand sample as shown in Figure 2.</li>
	</ol>
	</li>
	<li>Preparation of hollow brick mold 
	NOTE: The hollow brick mold includes a rigid holder, a flexible layer, and cardboard tubes. The size of the cardboard tube is 60 mm x 140 mm x 60 mm. Three chambers are included in one mold and the size of each mold chamber is 177.8 mm in length, 76.2 mm in width and 38.1 mm in height in this procedure.
	<ol>
		<li>Prepare the polypropylene perforated sheet for the constituent pieces of the rigid holder.</li>
		<li>Drill holes on the bottom of the rigid holder piece. The size of the holes is 61 mm in diameter. The location of holes in each chamber is shown in Figure 3a.</li>
		<li>Assemble the pieces of the rigid holder using plastic screws and nuts.</li>
		<li>Assemble the cardboard tubes in the drilled holes on the bottom of the rigid holder.</li>
		<li>Prepare the constituent parts of the geotextile flexible layer. The flexible layer consists of a bottom and a cover. Holes are also needed on the flexible layer at the same location of cardboard tubes.</li>
		<li>Once the sand is added into the mold, place the cover of flexible layer and fix by sewing on the top of the sand sample as shown in Figure 3b.</li>
	</ol>
	</li>
</ol>
4. Preparation of the batch reactor
NOTE: The reactor shown in Figure 4 consists of a plastic box, cementation media, a sample supported shelf, and air pumps. The soil samples can fully immerse into the cementation media while the cementation media can freely diffuse into the soil samples by this method. The air pump in the reactor provides oxygen for bacteria. To determine the effects of different oxygen supply on MICP treatment catalyzed by Sporosarcina pasteurii, Li et al. 201718 conducted contrast tests under three different conditions: an aerated condition, an air restricted condition, and an open-air condition. They found that a well-oxygenated condition is essential to improve MICP processes catalyzed by aerobic bacteria.
<ol>
	<li>Connect the air pump with air supply using a plastic hose.</li>
	<li>Place the air pump in the plastic box.</li>
	<li>Pour cementation media into the plastic box.</li>
</ol>
5. Preparation of soil samples
<ol>
	<li>Preparation of MICP-treated soil sample 
	NOTE: Ottawa sand (99.7% quartz) is used in the experiments. The sand is uniform with a median particle size of 0.46 mm and no fines are included. It is classified as poorly graded sand based on the Unified Soil Classified System (USCS).
	<ol>
		<li>Add dry sand into molds by the air pluviation method (FCFM, RFCM, hollow brick mold) to reach a median dense condition (Dr in the range of approximately 42&#8211;55%, and dry density of sand in the range of 1.58&#8211;1.64 g/cm3). 
		NOTE: The weight of sand varies according to different kinds of molds: 145 &#177; 5 g sand for the UCS test sample, which is 38.6 mm in diameter and 76.2 mm in height.</li>
		<li>Place the cover on the top of samples and fix it by sewing.</li>
		<li>Pour the bacteria solution with a fixed optical density value through the permeable geotextile cover into the samples and make sure that they are saturated. 
		NOTE: The amount of bacteria solution varied according to different samples: 50 mL of bacteria solution for a UCS test sample, which is 38.6 mm in diameter and 76.2 mm in height.</li>
		<li>Place samples on the sample supported shelf as shown in Figure 5a.</li>
		<li>Immerse the entire shelf into the batch reactor filled with cementation media.</li>
		<li>Turn on the air supply and adjust the air output to keep 100% air saturation. Wait for 7 days of MICP reaction.</li>
		<li>Take out the samples from the reactor as shown in Figure 5b.</li>
		<li>Remove the samples by cutting the full contact flexible mold or demolding the rigid holder and then cutting the flexible layer.</li>
		<li>Wash the samples with water to remove the residual solution in the pore space.</li>
		<li>Place the samples in the 105 &#176;C oven for 48 h until their weights remain constant. The samples can be tested or treated additionally after the oven drying.</li>
	</ol>
	</li>
	<li>Preparation of fiber reinforced MICP-treated soil sample 
	NOTE: Synthetic fiber (see Table of Materials) and natural palm fiber as shown in Figure 6 are used in these procedures.
	<ol>
		<li>For the synthetic fiber, mix the proposed content of fibers and 900 g of dry sand in small increments by hand to obtain a uniform mixture. The fiber content in this experiment is fixed as 0.3% by weight of the dry sand.</li>
		<li>For the natural palm fiber, distribute 760 g of sand into four equal parts. Add these four parts of sand and three layers of fiber in RFCM at intervals.</li>
		<li>Repeat the same procedure as steps 5.1.2&#8212;5.1.10 to get the MICP-treated sample.</li>
	</ol>
	</li>
	<li>Preparation of cement-treated bricks with bio-surface treatment 
	NOTE: Portland cement (TYPE I/II) with specific gravity of 3.15 is used as the cementing agent for the cement-treated samples in this experiment. The early strength gain of this cement allowed the various curing times ranged from 7 to 21 days. The proportion of added cement in this procedure is 10% by weight of dry sand.
	<ol>
		<li>Mix 900 g of sand, 90 g of cement, and 200 mL of water to achieve a uniform mixture.</li>
		<li>Add the mixture to the rigid mold. The size of rigid mold is 177.8 mm in length, 76.2 mm in width and 38.1 mm in height.</li>
		<li>Cure for 7 days at a constant humidity of 100% and constant temperature of 25 &#176;C.</li>
		<li>Place samples in the 105 &#176;C oven for 48 hours until their weights remain constant.</li>
		<li>Repeat the same procedure as steps 5.1.3&#8212;5.1.8.</li>
		<li>Place samples in the 105 &#176;C oven for 48 hours until their weights remain constant. The samples can be tested or treated additionally after the oven drying.</li>
	</ol>
	</li>
</ol>

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We have nothing to disclose.

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 effectively increased the penetration of cementation media into soil samples. The large amounts of pores of geotextile also allowed more precipitation occurring inside the mold to improve the strength of MICP-treated samples. The soil properties of MICP-treated samples, such as strength and calcite content, were greatly improved by using these molds in the immersion method. The immersion method showed an advantage in preparing prefabricated building materials, such as bio-bricks and bio hollow bricks. Synthetic fiber and natural fiber can be added in the soil to enhance the MICP-treated samples. Fiber addition is an appropriate way to improve prefabricated MICP-treated materials. MICP technique with immersion method can be applied to perform surface treatment for cement-treated bricks to improve their properties, such as enhancing the durability of cement-treated materials by reducing their permeability. However, this immersion method is difficult to implement in the field due to the limitation of its operation, future research on how to use this method on site is needed to apply this method in the field.

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, &gt;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, &gt;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, &gt;99%</td><td>Bio-world</td><td>41900068-3 (705727)</td><td></td></tr><tr><td>&lt;em&gt;Sporosarcina pasteurii&lt;/em&gt;</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, &gt;99.7%</td><td>Bio-world</td><td>42020309-2 (730205)</td><td></td></tr><tr><td>Urea, USP Grade, &gt;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 considered to be the mass of the carbonates precipitated in the MICP-treated samples. The CaCO3 content is indicated as percentage of sample weight. The CaCO3 content of the MICP-treated sample by the immersion method ranged from 9.0% to 9.5%. The results indicated that the precipitated CaCO3 was distributed uniformly throughout the soil sample. While Martinez et al. 201319 conducted experiments on 50 cm long sand columns by an injection method in the laboratory, they found that the calcite distributed nonuniformly along the MICP-treated sand column. Most of the calcite precipitated near the influent column and hindered the cementation reaction in the deeper section of the column.
The stress-strain curves of bio-brick reinforced with three layers of palm fiber and unreinforced bio-brick obtained using a four-point test is shown in Figure 8. The flexure strength of unreinforced bio-brick was 1,150 kPa, while that of reinforced bio-brick was 980 kPa. Their flexure strengths were similar, but the flexure strain was improved significantly by addition of the palm fiber. These results indicate that palm fiber can contribute to the improvement of ductility.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60059/60059fig01.jpg" /> 
Figure 1: Full contact flexible mold for direct shear tests. 
The full contact flexible molds were made of geotextile. The geotextile was a polypropylene, staple fiber and needle punched nonwoven material. The cylinder-shaped mold had a diameter of 62 mm and a height of 26 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60059/60059fig02.jpg" /> 
Figure 2: Sample preparation of bio-bricks. 
<img alt="Icon" src="/files/ftp_upload/60059/Circled_One.jpg" /> Assembled mold for brick; <img alt="Icon" src="/files/ftp_upload/60059/Circled_Two.jpg" /> Sand added into the mold; <img alt="Icon" src="/files/ftp_upload/60059/Circled_Three.jpg" /> Flexible cover added on the top of sand sample. The rigid full contact mold consists of a flexible layer and a rigid holder. The flexible layer was made of geotextile, and the rigid holder was made of a polypropylene perforated sheet. The mold consisted of three chambers and the size of each chamber was 177.8 mm in length, 76.2 mm in width and 38.1 mm in height. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60059/60059fig03.jpg" /> 
Figure 3: Cored brick molds. 
(a) Holes distribution on one chamber of mold; (b) Sample preparation of bio-cored bricks <img alt="Icon" src="/files/ftp_upload/60059/Circled_One.jpg" /> Assembled mold for cored brick; <img alt="Icon" src="/files/ftp_upload/60059/Circled_Two.jpg" /> Sand added into the mold; <img alt="Icon" src="/files/ftp_upload/60059/Circled_Three.jpg" /> Flexible cover added on the top of sand sample. The cored brick mold included a rigid holder, a flexible layer, and cardboard tubes. The size of cardboard tube was 60 mm x 140 mm x 60 mm. Three chambers were included in one mold and the size of each chamber of mold was 177.8 mm in length, 76.2 mm in width and 38.1 mm in height. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60059/60059fig04.jpg" /> 
Figure 4: Sketch of batch reactor for MICP. 
All samples were prepared in a completely stirred tank reactor. The batch reactor included a plastic box to contain soil samples and cementation media, a magnetic mixer to keep the solution uniform, and an air pump to provide oxygen for bacteria. A major feature of this method is to allow soil samples fully immerse into the cementation media and to allow the cementation media to freely penetrate into the soil samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60059/60059fig05.jpg" /> 
Figure 5: Soil samples placed on the supported shelf. 
(a) before the MICP reactions; (b) after the MICP reactions. The bio-brick samples were prepared with the full contact mold. A geotextile cover was applied on the top of the mold. Each bio-brick had a size of 177.8 mm in length, 76.2 mm in width and 38.1 mm in height. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60059/60059fig06.jpg" /> 
Figure 6: (a) Synthetic fiber; (b) natural palm fiber. 
The synthetic fiber was a homopolymer polypropylene multifilament fiber with a specific gravity of 0.91. It is chemically inert with high acid salt resistance. The length and thickness of the fibers used in this study were 12 and 0.1 mm, respectively, with an aspect ratio of 120 between the length and thickness of the fiber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60059/60059fig07.jpg" /> 
Figure 7: CaCO3 distribution in three areas of MICP-treated sample. 
Three zones were divided in the sample. In each zone, the amount of precipitated CaCO3 was measured and calculated as a percentage by weight. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60059/60059fig08.jpg" /> 
Figure 8: Flexure stress as a function of flexure strain for unreinforced bio-brick and palm fiber reinforced bio-brick with MICP treatment. 
The flexure strength of unreinforced bio-brick was 1,150 kPa, while that of reinforced bio-brick was 980 kPa. The flexure strain was improved significantly by addition of palm fiber. These results indicate that palm fiber can contribute to the improvement of ductility. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60059/60059fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Chemical</td>
			<td colspan="4">Concentration of cementation media (g/L)</td>
		</tr>
		<tr>
			<td>0.25 M Ca</td>
			<td>0.5 M Ca</td>
			<td>1 M Ca</td>
			<td>1.5 M Ca</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Nutrient broth</td>
			<td>3</td>
			<td>3</td>
			<td>3</td>
			<td>3</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.12</td>
			<td>2.12</td>
			<td>2.12</td>
			<td>2.12</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>15</td>
			<td>30</td>
			<td>60</td>
			<td>90</td>
		</tr>
		<tr>
			<td>CaCl2&#8729;2H2O</td>
			<td>36.8</td>
			<td>73.5</td>
			<td>147</td>
			<td>220.5</td>
		</tr>
	</tbody>
</table>
Table 1: Chemical components of cementation media. The chemicals were used to prepare four concentrations of cementation media in 0.25 M Ca, 0.5 M Ca, 1 M Ca, and 1.5 M Ca. The urea-Ca2+ molar ratio was fixed as 1:1.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Engineering

9.4K Views.  Jackson State University. De onderdompeling methode aangetoond in dit protocol helpen onderzoeker verwijderen ongebroken MICP-behandelde bodemmonster voor mechanische testen door middel van verschillende schimmel. Deze techniek helpt MICP-behandeld bodemmonster krijgen meer uniforme neergeslagen calciumcarbonaat. Die MICP-behandelde bodemmonsters krijgen ook betere mechanische eigenschappen via deze methode.Het belangrijkste aspect van deze techniek is om ervoor te zorgen dat de activiteit van bacteriën goed g...

Video: Sandy Soil Improvement through Microbially Induced Calcite Precipitation MICP by Immersion

Investigation of Early Plasma Evolution Induced by Ultrashort Laser Pulses

Early plasma is generated owing to high intensity laser irradiation of target and the subsequent target material ionization. Its dynamics plays a significant role in laser-material interaction, especially in the air environment1-11.
Early plasma evolution has been captured through pump-probe shadowgraphy1-3 and interferometry1,4-7. However, the studied time frames and applied laser parameter ranges are limited. For example, direct examinations of plasma front locations and electron number densities within a delay time of 100 picosecond (ps) with respect to the laser pulse peak are still very few, especially for the ultrashort pulse of a duration around 100 femtosecond (fs) and a low power density around 1014 W/cm2. Early plasma generated under these conditions has only been captured recently with high temporal and spatial resolutions12. The detailed setup strategy and procedures of this high precision measurement will be illustrated in this paper. The rationale of the measurement is optical pump-probe shadowgraphy: one ultrashort laser pulse is split to a pump pulse and a probe pulse, while the delay time between them can be adjusted by changing their beam path lengths. The pump pulse ablates the target and generates the early plasma, and the probe pulse propagates through the plasma region and detects the non-uniformity of electron number density. In addition, animations are generated using the calculated results from the simulation model of Ref. 12 to illustrate the plasma formation and evolution with a very high resolution (0.04 ~ 1 ps).
Both the experimental method and the simulation method can be applied to a broad range of time frames and laser parameters. These methods can be used to examine the early plasma generated not only from metals, but also from semiconductors and insulators.

An experimental method to examine the early plasma evolution induced by ultrashort laser pulses is described. Using this method, high quality images of early plasma are obtained with high temporal and spatial resolutions. A novel integrated atomistic model is used to simulate and explain the mechanisms of early plasma.

Early plasma is generated owing to high intensity laser irradiation of target and the subsequent target material ionization. Its dynamics plays a ...

Real-Time DC-dynamic Biasing Method for Switching Time Improvement in Severely Underdamped Fringing-field Electrostatic MEMS Actuators

Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input bias voltage. However, the tradeoff for the improved switching performance is a relatively long settling time to reach each gap height in response to various applied voltages. Transient applied bias waveforms are employed to facilitate reduced switching times for electrostatic fringing-field MEMS actuators with high mechanical quality factors. Removing the underlying substrate of the fringing-field actuator creates the low mechanical damping environment necessary to effectively test the concept. The removal of the underlying substrate also a has substantial improvement on the reliability performance of the device in regards to failure due to stiction. Although DC-dynamic biasing is useful in improving settling time, the required slew rates for typical MEMS devices may place aggressive requirements on the charge pumps for fully-integrated on-chip designs. Additionally, there may be challenges integrating the substrate removal step into the back-end-of-line commercial CMOS processing steps. Experimental validation of fabricated actuators demonstrates an improvement of 50x in switching time when compared to conventional step biasing results. Compared to theoretical calculations, the experimental results are in good agreement.

The robust device design of fringing-field electrostatic MEMS actuators results in inherently low squeeze-film damping conditions and long settling times when performing switching operations using conventional step biasing. Real-time switching time improvement with DC-dynamic waveforms reduces the settling time of fringing-field MEMS actuators when transitioning between up-to-down and down-to-up states.

Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input ...

Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using visible light irradiation with plasmonic nanoparticles. The plasmonic nanoparticle is used to improve the reduction efficiency of GO. It only takes 30 min&#160;at RT by illuminating the solutions with Xe-lamp, the r-GO solutions can be obtained by completely removing gold nanoparticles through simple centrifugation step. The spherical gold nanoparticles (AuNPs) as compared to the other nanostructures is the most suitable plasmonic nanostructure for r-GO preparation. The reduced graphene oxide prepared using visible light and AuNPs was equally qualitative as chemically reduced graphene oxide, which was supported by various analytical techniques such as UV-Vis spectroscopy, Raman spectroscopy, powder XRD and XPS. The reduced graphene oxide prepared with visible light shows excellent quenching properties over the fluorescent molecules modified on ssDNA and excellent fluorescence recovery for target DNA detection. The r-GO prepared by recycled AuNPs is found to be of same quality with that of chemically reduced r-GO. The use of visible light with plasmonic nanoparticle demonstrates the good alternative method for r-GO synthesis.

A simple protocol for the preparation of reduced graphene oxide using visible light and plasmonic nanoparticle is described.

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Environment

Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with sufficient heavy metal adsorption capacity. To this end, blast furnace slag, a residue from iron-making, is utilized as the starting material. Calcium is selectively extracted from the slag by leaching with acetic acid (2 M CH3COOH) as the extraction agent. The filtered leachate is subsequently physico-chemically purified and then carbonated to form precipitated calcium carbonate (PCC) of high purity (&lt;2 wt% non-calcium impurities, according to ICP-MS analysis). Sodium hydroxide is added to neutralize the regenerated acetate. The morphological properties of the resulting calcitic PCC are tuned for its potential application as a filler in papermaking. In parallel, the residual solids from the extraction stage are subjected to hydrothermal conversion in a caustic solution (2 M NaOH) that leads to the predominant formation of a particular zeolitic mineral phase (detected by XRD), namely analcime (NaAlSi2O6&#8729;H2O). Based on its ability to adsorb Ni2+, as reported from batch adsorption experiments and ICP-OES analysis, this product can potentially be used in wastewater treatment or for environmental remediation applications.

A protocol for the parallel production of precipitated calcium carbonate and zeolitic material from blast furnace slag via mineral carbonation and alkaline hydrothermal conversion, respectively, is presented. The performance of the zeolitic material towards nickel adsorption is tested.

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with ...

Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in agricultural soils. To this end, the core soil samples taken at different depth (including 0-15 cm, 15-30 cm, and 30-60 cm profiles) are collected from an agriculture field, the topsoil of which has already been enriched with an alkaline earth metal silicate containing mineral (such as wollastonite). After transporting to the laboratory, the soil samples are air-dried and sieved. Then, the inorganic carbon content of the samples is determined by a volumetric method called calcimetry. The representative results presented herein showed five folded increments of inorganic carbon content in the soils amended with the Ca-silicate compared to control soils. This compositional change was accompanied by more than 1 unit of pH increase in the amended soils, implying high dissolution of the silicate. Mineralogical and morphological analyses, as well as elemental composition, further corroborate the increase in the inorganic carbon content of silicate-amended soils. The sampling and analysis methods presented in this study can be adopted by researchers and professionals looking to trace pedogenic inorganic carbon changes in soils and subsoils, including those amended with other suitable silicate rocks such as basalt and olivine. These methods can also be exploited as tools for verifying soil inorganic carbon sequestration by private and governmental entities to certify and award carbon credits.

The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Bioengineering

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living ...

Chemistry

Enhancing CO2 Sequestration: A Soil Biota Approach to Boost Mineral Weathering

Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies on accelerating the natural process of mineral weathering in soils by manipulating the abiotic variables that govern this process, in particular mineral grain size and exposure to acids dissolved in water. EW mainly aims at reducing atmospheric CO2 concentrations by enhancing inorganic carbon sequestration. Until now, knowledge of EW has been mainly gained through experiments that focused on the abiotic variables known for stimulating mineral weathering, thereby neglecting the potential influence of biotic components. While bacteria, fungi, and earthworms are known to increase mineral weathering rates, the use of soil organisms in the context of EW remains underexplored. 
 This protocol describes the design and construction of an experimental setup developed to enhance mineral weathering rates through soil organisms while concurrently controlling abiotic conditions. The setup is designed to maximize weathering rates while maintaining soil organisms' activity. It consists of a large number of columns filled with rock powder and organic material, located in a climate chamber and with water applied via a downflow irrigation system. Columns are placed above a fridge containing jerrycans to collect the leachate. Representative results demonstrate that this setup is suitable to ensure the activity of soil organisms and quantify their effect on inorganic carbon sequestration. Challenges remain in minimizing leachate losses, ensuring homogeneous ventilation through the climate chamber, and avoiding flooding of the columns. With this setup, an innovative and promising approach is proposed to enhance mineral weathering rates through the activity of soil biota and disentangle the effect of biotic and abiotic factors as drivers of EW.

Author Spotlight: Unraveling the Role of Earthworms in Enhancing Mineral Weathering for CO2 Removal 

Here we present the construction and operation of an experimental setup to enhance mineral weathering through the activity of soil organisms while concurrently manipulating abiotic variables known to stimulate weathering. Representative results from the functioning of the setup and sample analyses are discussed together with points for improvement.

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies ...

Biochemistry

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

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

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