To allow WB800 function properly in concrete to fill the crack, we have to ensure that bacterium remain as sporse – a stable and dormant state in which it will remain uninterrupted by other factors until the cracks appear. Thus we need to insulate them from outside environment. Under this consideration, we designed a kind of micro-capsule composed of Microcrystalline Cellulose material sheltering the spore from outside. MCC-short for microcrystalline cellulose-is a stable and non-toxic composite material, keeps spores in isolation from outer environment. In the meantime, use of micro-capsule can enwrap the spores, thus promoting the spores’ concentration in each unit volume and enhancing the repairing effect. So we designed the micro-capsule to embed bacterium as spores in concrete properly.
Team:SZU-China/Design
DESIGN
As we have described in the background, a self-repairing device is required to protect steel bars in concrete, and to achieve such a goal, we can embed genetically modified Bacillus subtilis inside concrete.
To ensure that bacteria can function properly in concrete, we first improved alkali resistance ability, germination ability and mineralization capacity of Bacillus subtilis significantly. Then, we used a special technique to make microcapsules containing spores of Bacillus subtilis, along with necessary nutrients for germination. The microcapsules are incorporated into concrete of a certain proportion.
When microcracks inside buildings appear, the microcapsules will be torn apart and water is infiltrated to dissolve the nutrient. Spores are stimulated by mutrients into germination state and finally return to normal activity. Carbonic anhydrase of Bacillus subtilis promotes CO2 hydration to produce CO32-, which binds to free Ca2+ in the environment to form calcium carbonate deposits, thereby enabling microcracks to self-repair. As a result, microcracks are isolated from the external environment, preventing further corrosion due to internal reinforcement.
We chose Bacillus subtilis this year as the chassis due to its ability to form spore, which enables the bacteria to lie dormant for extended periods and protect itself from harsh environment, for instant extreme high or low pH, high temperature, and desiccation. In all the strains of Bacillus Subtilis, B.S168 is the most common one. However, since Bacillus Subtilis of this strain secretes digestive enzymes, which interrupt exogenous gene expression, we chose a modified strain – WB800, which has no digestive enzymes.
To activate germination ability of spores
As mentioned above, the embedded spores remain dormant in the micro-capsule. In order to wake them up, we transferred the gene, gerA, into our bacteria and overexpressed it. This gene encodes the nutrient receptor on the inner membrane, which can detect the outer nutrient substance, for example L-alanine, and activate germination and initiate our system through complex signaling pathways.
To resist certain alkaline environment
Considering the working circumstance of our system, concrete, where pH level is rather high, we need to enhance the alkali resistance ability of our bacteria. Thus we transferred two alkali resistant genes.
1. The expression of gene tupA results in a kind of enzyme which can shift glucuronic acid and L-glutamic acid into polyglucuronic acid and poly-γ-L-glutamic acid. These polymers will form a layer of protection barrier on the cell wall and neutralize the extracellular hydroxyl.
2. In the meantime, we transferred the second alkali resistant gene, nhaC. It is the coding gene for proton pumps on the cell membrane. These proton pumps play a key role in regulating cytoplasmic pH valued by coupling net hydrogen ions in and sodions out simultaneously, thus can balance the endocellular pH.
To produce CaCO3 for crack-filling
The key point of self-repairing system is to allow our bacteria producing calcium carbonate for crack-filling. To achieve this goal, we transferred the gene of carbonic anhydrase into our bacteria. This gene produces highly efficient enzymes that catalyze the reversible reaction of hydration of CO2 and form bicarbonate and protons rapidly. bicarbonate radicals will diffuse out of the cell along with the concentration gradient and combine with the hydroxyl and calcium iron in the concrete environment, which will become calcium carbonate sediment. Now the calcium carbonate can go and fill the micro cracks.
Here is a video for the specific mechanism described above.
[1] Tjalsma H, Bolhuis A, Jongbloed J D, et al. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome[J]. Microbiology & Molecular Biology Reviews Mmbr, 2000, 64(3):515.
[2]沈卫锋, 牛宝龙, 翁宏飚,等. 枯草芽孢杆菌作为外源基因表达系统的研究进展[J]. 浙江农业学报, 2005, 17(4):234-238.
[3]Løvdal I S, From C, Madslien E H, et al. Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores[J]. Bmc Microbiology, 2012, 12(1):1-12.
[4]Ito M, Guffanti A A, Zemsky J, et al. Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4[J]. Journal of Bacteriology, 1997, 179(12):3851-3857.
[5]https://en.wikipedia.org/wiki/Microcrystalline_cellulose
