Team:UCSC/Purification
Purification
Context
Our bioreactor is a chemostat, which presents a difficulty in filtration. Raw nutrients are constantly pumped into the bioreactor as existing liquid is constantly pumped out, containing water, sugars, bacteria, and erythritol. Filtering live, and actively growing, bacterial cells with a simple microfilter will result in the formation of a biolayer on the filter surface as the bacteria are trapped but continue to grow. The biolayer would prevent liquid from passing through, rendering the filter useless. The microfilter would need to be changed many times and would be an inefficient method of filtration. Remaining sugars present in the erythritol solution creates a lower level of purity when the erythritol is crystallized, and could have adverse effects on the ease of crystallization. Batch reactors avoid this issue because all of the initial sugar is consumed, and the yeast, bacteria or other reaction agent can be easily killed before filtration.Purpose
The purpose of the experiments within the purification team was to design, optimize, and quantify the efficiency of a filter or filtration method. In the scope of the IGEM project, this team is responsible for creating a method in which the live bacteria is removed and erythritol is separated from the other sugars. In addition to this, we quantified the lethal concentration of erythritol in solution for B. subtilis.
Prototype evolution! From left to right: prototype 1, prototype 2, prototype 1&2 in series, prototype 2 repaired, and prototype 3.
Bacterial trial plate evolution! From left to right: prototype 1/trial 1, prototype 3/trial 2, prototype 2/trial 2, prototype 1'/trial 2, and prototype 3/trial 1. The bacterial reduction power generated from each filter is displayed below each plate respectively, as a percentage of the total number of bacteria removed. The bar chart below sums up the number of colonies found on each plate.
Flowchart of the overall purification process. Ion exchange resins and hot plate images provided by Dartmouth and Eurodyne respectively.
Filter Design
The bacterial filter we designed has a two chamber PVC body and two diatomaceous earth layers. The filter is designed to be placed upright for use of the diatomaceous earth as well as using gravity to assist in the flow rate. Both chambers are 6 inches and have an inner diameter of 1.5 inches. The diatomaceous earth was held in place by a silk disk and a glass fiber filter. The top layer contains 4g of diatomaceous earth, and the bottom layer contains 2.5g. The bacterial reduction power was tested to be 1x10^9. The diatomaceous earth layers would have to be changed at intervals. However, such a system could be put in place to divert flow into one PVC body while the other is being changed, so that the flow is not interrupted.
Results from the third trial that supported a bacterial reduction power of 1x10^9 for our final filter.
Overall Design
The initial concept of the procedure included a first step that let the bacteria continue to consume sugars that exited the chemostat in solution and a final step that separated the erythritol from the remaining sugars in solution. The first step has not been tested because the final microbe is not available for characterization yet. The length of the tubing can be adjusted to allow the bacteria to consume the most sugar possible so that more erythritol is produced and less separation is required. This tubing will feed into the bacterial filter that has been designed and characterized. The bacterial filter will then feed into a holding vessel prior to a column containing ion exchange resin beads.
Ion exchange resins are used to separate dissolved substances in solutions. Currently, it is used in the sugar industry to chromatographically separate and purify separate components of a sugar solution. The ion exchange resins create a chromatographic separation by forming weak ligand bonds between the substances in solution and ions held in the beads. These interactions have varying strengths and release the different substances at different times. This creates the compound separation. High fructose corn syrup is produced using an ion exchange resin system that separates glucose from fructose and remixes the sugars in a different ratio to achieve 55% fructose (DOW technical manual). DOW produces a line of ion exchange resins solely for sugar and polyol purification. A strong acid cation exchange resin, DOWEX MONOSPHERE 88H, was identified to be the exchange resin that the purification team will investigate. Characterizing the flow and obtaining the erythritol portion are the last steps to be investigated in the purification process.
