Overview

The data presented on this page reveals that bricks made with E. coli INP-sil in TB-Si solution, which promotes silica polymerization, were visually stronger than traditional urea/calcium cement bricks, though still weaker than the positive control. Attempts to fortify these bricks with calcium and carbonate ions improved strength but did not match the control. This data allowed the team to shift focus towards using biosilicification for agricultural and dust mitigation purposes.

_{Figure 1: Condensation of two orthosilicate molecules with the elimination of water.}

Original Project Aims

Research has uncovered the process of producing biocement through microbiologically induced calcium carbonate precipitation (MICP) (Iqbal, Wong, & Kong, 2021). MICP is a biochemical mechanism that includes hydrolysis (decomposition of molecules via water) to promote urease enzymatic activity by microbes such as Sporosarcina pasteurii (S. pasteurii) that yield carbonate (CO₃^2-) ions with ammonium (NH⁴⁺) as a byproduct. Though this method produces a consistently uniform and strong cement, the ammonium byproduct poses a significant environmental risk. If applied to an agricultural setting, the high levels of ammonium in the soil can lead to interveinal chlorosis, inhibition of root growth, necrosis of leaves, and many other botanically fatal effects (Guan, 2016). The introduction of a new mechanism, biosilicification, has the potential to address the same criteria that makes an organically produced cement attractive, with the added benefit of a more conscious environmental impact. Biosilicification involves genetic modification of Escherichia coli (E. coli), to express the silicatein-α gene which catalyzes the polymerization of silica in the applied soil, sand, or substrate. When supplemented with sodium orthosilicate, the silicatein-α enzyme will bind soil/sand/substrate particles together, creating a unified aggregate, or Bio Si-ment.

Cycle 1: Testing the Effect of Removing Urea

Design

The first experiment aimed to evaluate whether the removal of the ammonia-producing ingredient, urea, in the biosilification process would affect the structural integrity of bricks. The hypothesis was that genetically engineered E. coli expressing the silicatein-α gene could maintain the strength of the bricks without producing ammonium, eliminating the need for urea in the biosilification process.

Build

We created four test groups:

Three bricks with 100% Urea/Calcium Solution.
Three bricks with 100% TB-Si Solution (Terrific Broth with 2mM Sodium orthosilicate) (no urea).
A positive control using S. pasteurii (traditional biocementation bacteria).
A negative control with wild-type E. coli (without the INP-silicatein-α gene).

Test

Qualitative results indicated that the 100% TB-Si Solution produced bricks closest in structural integrity to the positive control (Figure 2 & 4). This suggests that removing urea did not significantly compromise the brick's strength. Meanwhile, the 100% Urea/Calcium Solution bricks performed worse than the 100% TB-Si Solution bricks in maintaining structural integrity.

_{Figure 2: 100% TB-Si Solution}

_{Figure 3: 100% Urea/Calcium Solution}

_{Figure 4: Right: Positive Control (S. pasteurii) and
Left: Negative Control (E. coli w/o INP-sil)}

Cycle 2: Investigating the Effects of Carbonate and Calcium Additives

Design

Based on the success of the 100% TB-Si Solution in the first cycle, we designed a second experiment to test whether adding carbonate or calcium would strengthen the 0% cement bricks and improve their robustness through the formation of calcium carbonate in addition to biosilicification.

Build

We built two new test groups for this cycle:

Bricks with 100% TB-Si Solution fortified with carbonate.
Bricks with 100% TB-Si Solution fortified with calcium.

Test

Results showed that the carbonate-fortified bricks (Figure 7) were stronger than those fortified with calcium alone (Figure 6). The carbonate bricks were able to stand on their own, similar to the positive control, while the calcium-fortified bricks were less stable. These results confirmed that carbonate increased the strength of the 100% TB-Si Solution bricks.

_{Figure 5: Right: Positive Control (S. pasteurii) and
Left: Negative Control (E. coli w/o INP-sil)}

_{Figure 6: 100% TB-Si and Calcium Solution}

_{Figure 7: 100% TB-Si and Carbonate Solution}

Cycle 3: Refining the Experiment by Removing Mesh and Using Lubricants

Design

With promising results from the previous cycles, we wanted to gather more data to further support our hypothesis that the 100% TB-Si Solution was stronger than the 100% Urea/Calcium Solution. To mitigate the crumbling of sand particles observed in earlier tests, we designed a third experiment in which we removed the mesh and used Vaseline as a lubricant for improved cohesion.

Build

This cycle mirrored the first iteration’s brick construction:

Bricks with 100% Urea/Calcium Solution.
Bricks with 100% TB-Si Solution, but with the modifications of removing mesh and using Vaseline as a lubricant.

Test

The qualitative data from this iteration once again demonstrated that the 100% TB-Si Solution bricks (Figure 9) were stronger than the 100% TB-Si Solution. The Vaseline-lubricated bricks showed improved structural integrity, and the removal of mesh reduced the issue of crumbling. These results confirmed that the 100% TB-Si Solution produced more robust bricks than the 100% Urea/Calcium Solution under these conditions.Furthermore, we were able to collect quantatative data on these bricks using a fine pressure gauge. As seen in Figure 11, the E. coli INP-sil TB-Si solution produced significantly stronger bricks than the 100% Urea/Calcium Solution and negative control.

_{Figure 8: 100% Urea/Calcium Solution}

_{Figure 9: 100% TB-Si (no Urea) Solution}

_{Figure 10: Left: Positive Control (S. pasteurii) and
Right: Negative Control (E. coli w/o INP-sil)}

Describe what your results mean

_{Figure 11: Statistical Analysis of Brick Strength}

The 2024 iGEM team's project inspiration goes all the way back to our 2022 team. In 2022, the team worked on incorporating the silicatein-α gene from certain sea sponges to enhance the UV protection of S. pasteurii bricks. The implementation of this gene in biocementation showed promise, leading USAFA to partner with the University of Virginia to further explore its capabilities. The silicatein-α gene was modified with an Ice Nucleation Protein (INP) and then tested with the same cement solution used with S. pasteurii. When both types of bricks were tested, the compressive strength test revealed that, although the INP-silicatein bricks were weaker than the positive control, S. pasteurii, the INP-silicatein gene was still a viable option for biocementation.

The main concern remaining from the research was whether the ammonium byproduct from the chemical process could be eliminated. The USAFA iGEM team of 2024 assessed the situation, tested, and adjusted our brick-making process from 2022. We focused on growing INP-silicatein-α (INP-sil) E. coli edited bacteria in sand without urea/calcium cement solution and comparing it to sand with urea/calcium cement solution.

The results displayed when INP-sil expressing E. coli was paired with TB-Si cement solution (which provides additional bacterial growth medium and the precursors for silica polymerization) it produced the more visually structurally sound brick compared to the traditional ammonium producing urea/calcium cement solution. This remained consistent for all cycles of brick testing.

In an effort to match the TB-Si bricks strengths to that of the positive control, we aimed to fortify the TB-Si bricks. Thus, we added calcium ion and carbonate ions solutions to the bricks to test if this fortification would increase the strength to match that of the positive control. We visually observed that the carbonate dose was more structurally sound than the calcium dose; however, the carbonate fortification was still weaker than the positive control.

With bricks that still crumbled upon extraction from molds, our team wanted to see if Vaseline lined molds enabled the bricks to maintain their structural integrity. The testing of this variable was not very successful, however, we were able to gather more results comparing TB-Si solution to the urea/calcium solution. Each TB-Si solution appeared visually to be stronger than the urea/calcium solutions.

Visual displayed that TB-Si did indeed perform cementation via biosilicification; however, the feasibility of creating biosilicification bricks with strength comparable to the positive control was limited as seen in quantative data (Figure 11). Thus, our team then shifted its focus from manufacturing bricks to exploring how the expression of silica could benefit soil for agricultural communities in promoting plant germination, soil erosion, and dust mitigation.

Our team was able to pass off our discoveries to the dust mitigation team. They found that S. pasteurii with urea/caclium solution did not promote plant growth, whereas the INP-sil with the TB-Si solution did promote germination. This testing significantly impacted our project, and the flexibility we exercised provided a valuable solution to an existing problem. Our team has certainly embraced the design, build, and test framework while remaining adaptable throughout the process.

Project Achievements

Successful Results

TB-Si as a Viable Alternative: As seen in Figure 11, TB-Si (biosilicification-promoting) solution produced significantly stronger bricks compared to urea/calcium-based solutions across multiple test cycles.

Unsuccessful Results and Lessons Learned

Vaseline Tests: Attempting to apply Vaseline inside the mold walls to facilitate easier extraction of bricks was not successful.
Limited Statistical Analysis: The Bricks team spent two months making bricks without results that were structurally sound enough to test using a hydraulic press. Thus, we had to rely on visual data and a pinpoint pressure guage to measure strength of the bricks. With these data, we acknowledged that, due to the vast and significant difference between brick strength of biocemented S. pasteurii bricks and the biosilicificied INP-sil E. coli bricks, producing biosilicified bricks was not feasible on our timeline. Thus, the data found from the trials above inspired the application of biosilicification for dust mitigiation, prevention of soil erosion, and promotion of plant growth.