Team:SZU-China/Demonstrate
Demonstrate
We want to know to what extent does the recombinant B. subtilis can heal the concrete. And doing all this experiment (shown previously in Results can only support the B. subtilis in speculation. But we don’t know yet. So we take a step forward, establishing a concrete-model to understand the incredible interaction between concrete and the modified B. subtilis. To achieve this, the first question would be about the endospores. We want to create a interactive way, where the endospores can “feel” the microcrack and active itself to repair the concrete.That also means, the endospores would never germinate until microcrack appears. We developed microcapsules embedding method to achieve this. (As shown below)
After some days for the drying of concrete mold. Take out the column-like concrete from the mold. Then spilt the concrete right through the sided itches, exposing the microcapsule. Now if we put the two parts of concrete column together by sticking them with a tape, we can build a micro-crack on the upper surface. So we mark these concrete columns and put them into the artificial seawaters for 28 days. And you find the micro-crack on the upper surface be repaired like they never ever existed (as shown below)
Spores (whole yeast medium and 2SG medium) were collected on a solid medium for 7 days.（As shown below)
2 ml of Deionized water was added to the solid culture dish and the spores were scraped with an applicator. The spore suspension was poured directly into a 10 ml centrifuge tube, and each group collected spores of 4-5 solid dishes
Microscopic microscopy spore detection: add deionized water on solid medium, scrape the spores, take the suspension 5μL on the slide, cover the coverslip under inverted microscope microscopy, start from the low magnification of the microscope, adjust the fine focus screw, and access the image in phase difference mode
The spore suspension was centrifuged at 8000 r / min for 2 min, and the supernatant was discarded. To the precipitate, deionized water was added to 8 ml and the mixture was washed 8 times(As shown below)
Configure CHL nutrient solution: 25 g of sodium lactate, 5 g of sodium nitrate, 0.0075 g of potassium dihydrogen phosphate, 0.375 g of magnesium sulfate, 1.25 g of inosine (last step). Add sodium lactate (viscous liquid), add the rotor to start mixing and then weighing the other components were added to the sodium lactate until completely mixed, and then 50ml volume to 50ml.
Dilute the spores with CHL nutrient solution and count with a microscope plate under inverted microscope (cover-slide covers half of the counting chamber, take 2μL of spore CHL suspension on the coverslip boundary, push the coverslip to make bacteria Liquid into the counting room)
Weigh 1gMCC and 0.1g clay, mixed in a clean beaker, add 1.5ml spore CHL suspension (3 times to join, each 0.5ml) mixed evenly. Take a hand to do a handful of microcapsules: first with four fingers from four directions to strengthen the powder, and then gently kneaded into a small ball, put in front of a good wrinkled weighing paper culture dish, cover the lid , Placed in 37 degrees incubator in 60h for condensation of water
After 60h, use the culture dishes covered with cement powder, sieve to remove the coarse pieces of coarse cement powder 200g, according to the 0.4 water: cement ratio by adding 80ml deionized water, installed stir the machine, storing form the low-speed mixing 1min, and then turn to the highest Speed stirring for 2 min.
Remove the cement basin, fill the cylindrical hollow resin into the cavity of the test chamber into the two-thirds of the cement, into a microcapsule on both sides of the gap in the middle, and then fill the cement ramming test block
Gathered all concrete mold together, covered with a layer of plastic wrap, mark the mark, conservation for 2 days
After that, take the solidified cement test block from the resin mold out, the cement block into the bag, make a mark, sealed 2 days
Split the cement in the bag test block, with a vise to the cylindrical cement test block from the side of the split, to collect the appropriate split test block (buried in the test block in the microcapsules were split into two halves), with a transparent tape The pieces of the test pieces were re-bonded into a cylindrical shape
Put the re-glued test piece into the clean beaker vertically, pour it into the artificial seawater solution until the liquid level has not been tested. Place a layer of gauze on the beaker and place it in the shade for 28 days. Add artificial seawater to prevent evaporation
After 28 days, remove the test block and place the test block under microscopic examination of the microfracture
As shown in Fig 1, under the microscope, you can see the microcrack lying in the middle between two grooves of concrete mold. And this is how it looks like when we have a x1000 horizon after 12days. So it is obviously that the microcrack has been repaired under the self-healing reaction of recombinant B.subtilis.
We collected the particles that filled the cracks, and the energy spectrum analysis confirmed the calcium carbonate composition (shown in Fig 2). In other words, it works.
Reference
[1]PENGHuia, ZHANGJinlong, LIUBing, DENGXua, XINGFeng. Development of microbial self-healing technique in concrete[J]. concrete,2014,(08):38-42+48.
[2]Zhang JinLong, Wang ChaoGang,Wang QinLiang, Deng Xu*, Liu Bing,Han Ningxu, Xing Feng (2016). A binary concrete crack self-healing system containing oxygen-releasing tablet and bacteria and its Ca2+-precipitation performance. Applied Microbiology and Biotechnology.2016, doi:10.1007/s00253-016-7741-z
[3]Zhang Jinlong, Wu Rongshan, Li Yanmei, Zhong Jiayan, Deng Xu*, Liu Bing, Han Ningxu, Xing Feng. Screening of bacteria for self-healing of concrete cracks and optimization of the microbial calcium precipitation process. Applied Microbiology and Biotechnology. 2016, 100(15): 6661-6670.
