Experimental Design: Agricultural Applications

The effects of dust and unstable soil permeate agricultural environments, threatening crop yields and endangering the livelihood of farmers across the globe. Because of its theoretically environmentally friendly nature, biosilicification is a promising method to stabilize soil and reduce soil loss. Literature has even shown that biocemented soils have reduced water evaporation, beneficial in minimizing the effects of drought (Xin-lun et al., 2024). Overall, biocement has shown promise in agriculture but cannot be used in high concentrations because of its harm to plants (He et al., 2023). Biosilicification has the potential to provide the benefits of biocementation while minimizing environmental harm, making it effective in agricultural applications.

Cycle 1: Testing Effects on Plant Growth

Design

Biocementation and biosilicification both have complex chemical pathways that introduce several chemicals into treated soil. In particular, biocementation introduces ammonia into the soil as a byproduct of microbially induced calcium carbonate precipitation. Biosilicification offers a more environmentally friendly approach, producing silica-based bonding to solidify soil. To test the effects of both processes, Arabidopsis was chosen due to its hardy nature. The goal of this experiment was to see if there was a difference in growth between biosilicified, biocemented, and untreated groups.

Build

Experimental groups included a pre-cemented group cemented and mixed as preprepared soil, one cemented immediately after seeds were planted, and one cemented after germination. Each experimental group was tested with 3 iterations. All groups were given 10 ml of water each day.

Test

After initial planting and testing, it became clear relevant data would come too slowly. Arabidopsis typically takes 13-15 days to germinate, making collecting data relatively slow, particularly for post-germination experimental groups. Additionally, in the time since we initially planted and treated the soil with a similar protocol to brick making, other experiments revealed that TB-Si broth was far more effective for silicification in the modified E. coli INP-sil. As a result, we looked to perform another experiment with updated biosilicification solutions.

Learn

Shortly after planting the Arabidopsis, we determined that to run several iterations we needed a faster germinating seed. As a result, we started researching plants with faster germination rates and came across the Wisconsin Fast Plant,a rapid-cycling variety of Brassica, which has a germination rate of 3-5 days. In order to improve the efficiency of testing, we began an experiment nearly identical to the Arabidopsis one.

Cycle 2: Faster Growing Plants with Updated Solutions

Design

Similar to the Arabidopsis experiment, initial testing was designed around finding the effects of both biocementation and biosilicification at different times in the germination process. Experimental groups included a pre-cemented group cemented and mixed as pre-prepared soil, one cemented immediately after seeds were planted, and one cemented after germination. The goal of this experiment was to gather data on whether either biosilicification or biocementation affected the germination rate of plants, as well as when either process could be implemented into the agricultural process.

Build

For this experiment, to minimize the space needed for plant growth in our small workspace we decided to use small test tubes as planting pots. Because Wisconsin Fast Plants do not require large amounts of soil, we believed this would be a good solution in order minimize recources. The same experimental groups were planted and watered with 10 ml each day.

Test

After initial solution was applied and each plant was watered it became clear that liquid was not evaporating as we expected. After the first day of watering water and biocement/biosilification solutions remained in each test tube. In an attempt to not drown the plant's, liquid was pipetted off of the top of each test tube. Despite this, the seeds still struggled to germinate in the experimental groups because they had been immersed in bacterial solutions, or because they had drowned. Our team decided that our experiment needed to be updated with a new drainage solution so seeds did not drown.

Cycle 3: Using larger planting pots with drainage.

Design

As we began Cycle 3 we decided to use the same planting pots we had used in Cycle 1, largely because of the larger space and built in drainage. We also changed the experimental groups we tested. Both Cycle 1 and 2 showed little to no growth in either biocemented or biosilicificated soils. So instead of prioritizing on testing the effects of treatment at different germination stages we decided to test if plants could germination at all in bacterial solutions. We also decided to run 3 seperate soil samples for each group. These included a topsoil only group, a topsoil upper layer with zeolite beneath, and a 50/50 topsoil zeolite mixture. Each of these soil groups contained identical experimental groups.

Build

Using the larger planting grid from Cycle 1, a total of 54 seedlings were planted in 18 different experimental groups (6 per soil type).The experimental groups were all cemented/silicificated pre-germination, immediately after planting. Each was watered with 10 mL of water a day. Both E. coli INP-sil and H₂O-Si were tested for biosilicification experimental groups. After our team discovered that the bacterial cells surface-displaying the silicatein enzyme were capable of effective biosilicification when resuspended in water instead of their Terrific Broth (TB) growth medium, we switched to using water with sodium orthosilicate instead of TB-Si to minimize the chemicals/organic compounds being applied to the plants and thus decrease potential detrimental environmental impacts.

Test

After 3-4 days of consistent watering the first seeds began to germinate. The table shown below shows both the germination rate and average days until germination of each experimental group. Overall, this experiment yielded promising results as well as major takeaways from this experiment. Perhaps most promising is the germination within the E. coli H₂O-Si group within both the topsoil and topsoil/zeolite planting groups. It should be noted that there was very little growth in the 50/50 test group, potentially indicating that there were not enough nutrients in this soil mixture for plants to grow.

Project Achievements

Through 3 cycles, the agricultural applications of biosilicification look extremely promising. Cycle 3 yielded promising results, indicating that seeds are able to germinate in E. coli H₂O-Si. Although not germinating at the same rate and slightly slower than the water control, biosilicification groups were able to grow when no calcium-carbonate biocement groups germinated. This indicated that biosilicification is likely less harmful to plant growth. In the future, with the goal of applying dead bacteria with surface-displayed biosilicification enzyme rather than introducing live bacteria, biosilicification will likely be even less harmful to plant growth and germination.

Sources

Ji, X. L., Tang, C. S., Pan, X. H., Cai, Z. L., Liu, B., & Wang, D. L. (2024). Long‐term performance on drought mitigation of soil slope through bio‐approach of MICP: Evidence and Insight from Both Field and Laboratory Tests. Water Resources Research, 60(7), e2024WR037486.
He, J., Liu, Y., Liu, L., Yan, B., Li, L., Meng, H., ... & Gao, Y. (2023). Recent development on optimization of bio-cementation for soil stabilization and wind erosion control. Biogeotechnics, 1(2), 100022.