Preliminary trials of testing for phosphorus using the lachat and the ICP-OES at the USDA National Soil Erosion Research Laboratory began. Both machines operate via spectrophotometry, comparing measured values to standard curves. The lachat measures orthophosphate, or soluble reactive phosphorus, while the ICP measures total phosphorus, the entirety of phosphorus contained in a sample. Corresponding with this endeavor, many bottles of Tris-HCl buffer, the media in which phosphorus-eating E. coli will be suspended, were created and autoclaved for future use.




The week continued by learning to make methanol-free bioencapsulation beads using the sol-gel method. A graduate student in the lab worked with team members to describe and demonstrate the protocol. After practice forming the beads, the team switched to a different method that could be more easily controlled and manipulated. E. coli were added to the beads to determine whether or not they would be able to survive within the structure.



Fluorescent Beads 1


Above: Shots of silica beads containing E. coli expressing green fluorescent protein, GFP, (left) and red fluorescent protein, RFP, (right) imaged under EVOS fluorescence microscope; the circled areas on the left indicate particularly bright spots where it can be reasonably inferred that a clump of E. coli is successfully producing GFP



Silica Beads 2


Above: Additional images of silica beads with E. coli expressing RFP (left) and GFP (right); the brightly-glowing RFP beads indicate RFP expression




Based on results from the lachat and ICP and the discovery that E. coli would not grow in the originally proposed Tris-HCl buffered solution, the measurement of phosphorus uptake protocol was modified after a productive meeting with advising scientists. Modified microbes will now be suspended in a minimal media solution whose components can be processed by both the sensitive lachat and ICP instruments. Prototype modeling progressed, with team member Barrett composing a 3D model to better-depict uptake system hardware. In the afternoon, a few team members also visited the West Lafayette Wastewater Treatment facility, where they learned about the local area’s methods for phosphorus removal featuring their newly-implemented chemical system.



3D BioreactorAbove: Current bioreactor prototype with 2 5-gallon buckets, an aquarium pump, connective tubing, and 3 standard water filter chambers to house E. coli



Additional trials of media and various water samples were run on both the lachat and ICP as team members were trained in their use. Colony PCR and sequencing to confirm the transformation of phosphorus genes into E. coli were also begun, with completion planned to coincide with the conclusion of next week.




Focus was placed on brainstorming possible development strategies of silica beads. Two components combine to make these beads: a sol phase and a buffer (Tris-HCl). The team has been trying to find the proper ratio of these two ingredients so beads solidify at an appropriate rate–too slowly, and an amorphous silica blob forms in the bottom of the mineral oil into which the beads drop; too quickly, and the mixture hardens inside the tubing through which it is dispensed. Cool temperatures slow the reaction, so the team has also tried completing the process in the cold room with mineral oil warmed on a hot plate, but to no avail. The current method involves the use of syringes to inject the two components into a small Y-shaped tube where they mix and then pass through a needle at the bottom to drop into mineral oil. According to calculations, 99,000 beads will be needed to fill a standard water filter canister; that’s a lot of drips!