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.
