Verifying biosilicification and measuring ammonium concentration
The data presented on this page suggests that E. coli INP-sil effectively facilitates biosilicification and the formation of amorphous aluminosilicate in aluminum-rich environments. Despite concerns about the efficacy of the ammonium test kit for bacterial test samples, the data suggest that E. coli INP-sil produces approximately 27.5x less ammonium than S. pasteurii, the standard bacterium used in biocementation.
In Sporosarcina pasteurii (S. pasteurii), MICP releases a high concentration of ammonia as a byproduct, posing potential problems in the application of biocementation (Carter, 2023). Ammonia is a compound which, when introduced into the environment, can be harmful to many organisms. This becomes a source of concern for real-world applications because of the potential for ammonia-contaminated runoff from biocement products. To effectively apply biocementation, this is a problem that is vital to solve. In the world of wastewater, extensive research has been done on cleaning up ammoniacal water sources (Adam, 2020). One common method has been the application of zeolites --- “aluminosilicate minerals with rigid anionic frameworks containing well-defined channels and cavities” (Pavelic, 2018). Due to these minerals’ ion-exchange properties, they have a great capacity to absorb ammonium, specifically in aqueous solutions. It has been suggested that biological platforms can be harnessed to naturally create aluminosilicates similar to zeolites. Currently, biosilification processes – being those that involve a cell-induced precipitation of silica structures – have been transferred from sponges to bacteria via a silicatein gene. Additionally, research done on diatom biosilification has suggested that the introduction of aluminum into the environment of a silica-producing organism could lead to formation of zeolite-like aluminosilicates (Kohler, 2017). While the biosilification of diatoms differs from that of sponges, it is proposed that introduction of aluminum in the environment of bacteria engineered with silicatein could result in the formation of aluminosilicate with absorption capabilities comparable to zeolites.
Description of INP:
Ice Nucleating Proteins (INPs) are naturally occurring proteins used in several species to facilitate ice formation. INPs naturally migrate to the outer surface of cells. By linking a protein in this case, silicatein-α, INPs act as transporter and have been shown to transport other proteins extracellularly (Kim and Ku, 2018). INP has a long history of successfully transporting enzymes to the outer cell membrane (Jung et al., 1998) making it a promising addition for our team.
Description of silicatein-α:
The silicatein-α gene is an engineered and truncated version of Suberites domunculas silicatein-α gene that has been codon optimized for E. coli. It was engineered by and gifted from collaborators at the University of Virginia. This gene produces the silicatein-α enzyme which acts as a scaffolding site to catalyze the biomineralization of silica spicules (Gao et al. 2024). In a lab setting, this enzyme has catalyzed the formation of calcium carbonate and silicon dioxide spicules (Natalio et al. 2013; Sadeghnejad 2019). Thus, our team has decided to use the silicatein-α gene for the biomineralization of orthosilicate to produce silicon dioxide. We have entered this truncated silicatein-α gene as a new part in the igem registry (BBa_K5199003)
Function of INP-sil:
Contemporary biocementation releases ammonium as a byproduct that leeches into the soil and poses potential environmental risks (Chuo et al. 2020, Esteban et al. 2016). The purpose of our INP and silicatein-α composite part is to function as an ammonium free alternative for biocementation. Silicatein-α serves as the biomineralization site, capable of creating an amorphous mixture of CaCO3 and SiO2 without the resulting ammonium byproduct (Natalio et al. 2013). INP functions as an extracellular transporter allowing the silicatein-α enzyme to be expressed on the external surface of E. coli where the biomineralization is occurring (Kim and Ku, 2018). Due to the passive nature of silicatein-α, this allows the enzyme to continue the biomineralization process even when the bacteria are dead, eliminating safety concerns of introducing living genetically modified organisms into the environment (Bawazer, 2013). This provides a safer and environmentally friendly alternative to contemporary biocementation that can be used for a myriad of applications. The INP-sil composite part is BBa_K5199004.
Design 1:
The objective was to create a biomineralization assay to verify and understand the production of CaCO₃ and SiO₂ under different conditions as well as create an amorphous aluminosilicate. After consulting with Dr. Knoerzer, we determined that IR spectroscopy would be the most efficient method for analyzing mineral production. Key test groups were identified:
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Design 2:
The objective remained to verify and understand the production of CaCO₃, SiO₂, and amorphous aluminosilicate. After addressing water interference issues in IR spectroscopy, the focus shifted to refining the assay with better dehydration techniques and testing for brick matrix formation using combined silicon dioxide and calcium carbonate production. Key groups tested:
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Results and Discussion:
After centrifuging the E. coli in Terrific Broth (TB), the supernatant was removed and replaced with water, showing that the cells could still polymerize in a water environment. Both E. coli INP-sil and E. coli wild type (WT) cells were resuspended in water along with various chemicals, following the growth and protein induction phases. Remarkably, the cells were able to perform biomineralization in water, and without the presence of TB. This explains why the plant and dust groups switched to water-Si instead of TB-Si, aiming to reduce possible ammonium content and prevent plant damage.
In the first iteration of the Infrared (IR) Spectroscopy testing, the pellets in Figure 1 were run and resulted in the same peaks/oscillations. Figure 1 does not match Figure 2, 3, or 4 standard IR spectrums, concluding that the first iteration of testing the pellets was overconcentrated with water which diluted the pellet to express H2O peaks. To overcome this issue, in the second iteration the pellets were spun down more to be more compact and then dried out to keep the water out of the IR spectrums. The results of the second iteration of testing are in the IR spectrum Figures below. The analysis of 1b, 2b, and 3b was carried out using IR spectroscopy. We completed these IR testing iterations and analyzed the spectrums with the aid of Dr. Knoerzer.
For IR analysis as shown in Figure 2 from the Synthesis of amorphous silica and sulfonic acid functionalized silica used as reinforced phase for polymer electrolyte membrane, only product 1b is present (Tran). There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm-1 to 500cm-1 signal the presence of Si-O-Si alone. All these peaks are indicative of a pure silicon dioxide product (1b).
For IR analysis as shown in Figure 3 from the National Institute of Standards and Technology, only the product 2b is present (NIST). The most prevalent functional group is carbonate (CO32-). It is shown with a sharp peak around 1500cm-1. In 2b, there is another strong peak around 1000cm-1, which displays a carboxylic acid OH bond on bicarbonate (HCO3-). These peaks are indicative of 2b, a pure calcium carbonate product.
For IR analysis as shown in Figure 4 from the Kinetic study of removal heavy metal from aqueous solution using the synthetic aluminum silicate, only product 3b is present (Treto-Suarez). There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peak from around 1100cm-1 signals the presence of Si-O-Si alone. Around the 500 to 1500cm-1 range shows the different types of peaks within the structures O−Si−O and Al−O−Si (Treto-Suarez). The most notable peak in this range is Al-O-Si around 500 to 1000cm-1. There are two peaks around 1630cm-1 to 1330cm-1, which is attributed to water within the Si-O-Si structure (Ellerbrock). These peaks are indicative of an amorphous hydrated aluminum silicate (3b).
For IR analysis as shown in Figure 5, it displays the product 1b and 2b. There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm-1 to 500cm-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b). Additionally, the product 2b is also present in Figure 4, with the most prevalent functional group carbonate (CO32-). It is shown with a sharp peak around 1500cm-1. In 2b, there is another strong peak around 1000cm-1, which displays a carboxylic acid OH bond on bicarbonate (HCO3-). These peaks are indicative of 2b, a pure calcium carbonate product. Figure 4 includes both the silicon dioxide and calcium carbonate as products on the IR spectra.
For IR analysis as shown in Figure 6, only product 1b is present. There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm-1 to 500cm-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).
For IR analysis as shown in Figure 7, only product 1b is present. There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm-1 to 500cm-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).
For IR analysis as shown in Figure 8, only product 1b is present. There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm-1 to 500cm-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).
For IR analysis as shown in Figure 9, only product 1b is present. There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm-1 to 500cm-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).
For IR analysis as shown in Figure 10, only product 2b is present. The most prevalent functional group is carbonate (CO32-). It is shown with a sharp peak around 1500cm-1. In 2b, there is another strong peak around 1000cm-1, which displays a carboxylic acid OH bond on bicarbonate (HCO3-). These peaks are indicative of Figure 3, a pure calcium carbonate product (2b).
For IR analysis as shown in Figure 11 from the Kinetic study of removal heavy metal from aqueous solution using the synthetic aluminum silicate, only product 3b is present (Treto-Suarez). There is an alcohol group at a broad peak around 3400cm-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peak from around 1100cm-1 signals the presence of Si-O-Si alone. Around the 500 to 1500cm-1 range shows the different types of peaks within the structures O−Si−O and Al−O−Si (Treto-Suarez). The most notable peak in this range is Al-O-Si around 500 to 1000cm-1. There are two peaks around 1630cm-1 to 1330cm-1, which is attributed to water within the Si-O-Si structure (Ellerbrock). These peaks are indicative of an amorphous hydrated aluminum silicate (3b).
Design 1:
The goal was to measure the initial ammonium concentrations in flowthrough samples from bricks made with:
This would allow us to assess potential environmental concerns by comparing the ammonium production of these different bacteria and understand how varying test conditions influence ammonium concentrations. We decided to use an ammonium test kit utilizing an enzyme coupled reaction and a plate reader for quantifying ammonium levels.
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The failure to generate a usable standard curve in all three trials strongly suggests that there might be a defect in the test kit, the protocol, or an issue with the plate reader. Potential sources of the problem could include:
Design 2:
The goal was to measure ammonium concentrations in flowthrough samples from bricks made with:
This would help assess potential environmental concerns by comparing ammonium production between these bacteria. An ammonium test kit with an enzyme-coupled reaction and a plate reader was selected for quantification.
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Ammonium Quantification:
As shown in Figures 13 and 14, the predicted ammonium concentrations for each dilution of the S. pasteurii and E. coli INP-sil dilutions was not consistent and showed some considerable variability. Ideally, each dilution would have an equal ammonium concentration when using the optical density in the standard curve and correcting for the dilution factor to determine the equivalent concentration of the undiluted test sample. The inconsistencies present in both the S. pasteurii and E. coli INP-sil dilutions suggests that the ammonium test kit used may not be an effective test method for our samples. Because the ammonium test kit relies on an enzyme coupled reaction for the colorimetric changes to occur, it is possible that the pH of the test samples is altering enzyme activity, the bacteria in the test samples produces a compound leading to partial inhibition of the enzymes, or the presence of bacteria in the test samples is leading to enzyme degradation in some way.
Although the data in Figures 13 and 14 suggests that the ammonium test kit provides erratic results when applied to bacterial test samples, the ammonium concentrations for average dilutions of S. pasteurii and E. coli INP-sil were compared in Figure 15. Using only dilutions with an optical density within the standard curve, an average ammonium concentration was calculated for the predicted undiluted test samples. These results suggest that there is approximately 27.5x as much ammonium produced by S. pasteurii than E. coli INP-sil. This matches our initial hypothesis because contemporary biocementation with S. pasteurii relies on the enzyme urease to break down urea and ultimately generates the production of ammonium ions. This enzyme is not present in E. coli INP-sil and helps explain the disparity between ammonium production.
Given the successful production of amorphous aluminosilicate as suggested by our IR data, a key next step will be to incorporate this material into brick production. We aim to assess how the inclusion of amorphous aluminosilicate affects both ammonium concentration in flowthrough samples and the overall strength of the bricks. This will help us understand its potential as a biocompatible and environmentally friendly material for construction applications. Additionally, we plan to test the effectiveness of zeolite in the brick matrix to determine its ability to absorb ammonium ions. Zeolites are known for their ion-exchange properties, and their inclusion in the flowthrough system may offer a novel way to mitigate ammonium release from biocementation processes, addressing environmental concerns associated with high ammonium concentrations. By comparing the ammonium levels and mechanical properties of bricks containing amorphous aluminosilicate, zeolite, or a combination of both, we will be able to optimize the material composition for environmental sustainability and structural integrity. These future tests will provide critical data for developing more sustainable, ammonium-reducing alternatives to traditional biocementation, enhancing both ecological impact and material performance.