Overview
After successful engineering of a high-yield Bacillus subtilis strain for ε-PLL biosynthesis, we focused on developing a robust downstream purification device. Through extensive design, build, test, learn cycles, we have established a user friendly purification system that delivers purified ε-PLL suitable for convenient integration into cling film manufacturing. This purified ε-PLL solution will ultimately be incorporated into our cling film product.
Cycle 1
Design
The main steps of purification involve adsorbing ε-PLL from the bacterial culture supernatant, desorbing the ε-PLL and further purification. Therefore, we design a 3-columned filtration system according to this process. In the first column, we fill in Amberlite IRC-50 to adsorb ε-PLL from the Bacterial culture supernatant. Different from other materials, it is easy to operate for absorption and elution, and has a high specificity.
| Material | Type | Advantage | Disadvantage |
|---|---|---|---|
| Amberlite IRC-50 | Weak acid cation exchange resin | High selectivity, food grade, reusable | Sensitive to pH (<5 reduces adsorption capacity) |
| Strong acid resin | Strong acid cation exchange resin | Strong capacity, wide pH range | Difficult desorption, requires strong acid/alkali, product prone to degradation |
The secondary and tertiary columns were packed with activated carbon to serve as polishing stages. This configuration effectively adsorbs residual pigments and volatile organic compounds that could affect the optical clarity and well functioning of the final cling film product.
| Material | Advantage | Disadvantage |
|---|---|---|
| Activated carbon | Cheap, suitable for lab operation | Small loss of ε-PLL, requires filtration |
| Macroporous resin | Reusable, high efficiency | Requires ethanol regeneration, higher cost |
The purification system utilizes air pumps to drive sequential processing of bacterial culture supernatant through three critical phases: (1) initial filtration, (2) ε-PLL elution, and (3) final polishing. All filtration columns are connected with silicon tubing. To streamline flow path switching during the ε-PLL adsorption/elution cycle on the primary Amberlite IRC-50 column, we integrated a three-way diverter valve at the column inlet. This configuration allows convenient transition between Supernatant loading and Acetic acid elution.
Build
The purification system was constructed by packing three purification columns filled with Amberlite IRC-50 cation-exchange resin (Column 1) and activated carbon (Columns 2-3) respectively. The columns were securely mounted on an adjustable scaffold and connected with silicone tubing. An air pump was then attached to the inlet terminal to drive fluid flow through the integrated purification cascade.
Test
To evaluate the mechanical properties of the device, we introduced water into the purification system and observed its flow. The device demonstrated excellent air-tightness, as evidenced by uninterrupted water processing in the connecting tube driven by the air pump.
Learn
Post-evaluation revealed several operational challenges requiring immediate attention. The system exhibited suboptimal processing speeds, indicating insufficient suction force from the air pump assembly. Concurrently, the column packing materials became suspended upon liquid introduction, causing visual obstruction of effluent monitoring. Furthermore, post-filtration analysis detected residual carbon particles in the filtrate, suggesting physical degradation of the column packing matrix. These findings collectively necessitate pump recalibration for enhanced flow rates, implementation of mechanical retainers to stabilize packing materials, and further filtration to remove remaining carbon particles.
Cycle 2
Design
To address the system inefficiencies found in the previous cycles, we implemented four targeted modifications: replacing the air pump with a high-flow peristaltic pump to enhance fluid processing rates, upgrading to larger-volume filtration columns to improve throughput capacity, installing spacer in all filtration columns to prevent packing material suspension, and incorporating a filtration cotton column to remove remaining carbon particles in the final solution.
Build
We change the device according to our design.
Test
Following resolution of the mechanical issues, we initiated productivity testing by loading 80 ml bacterial culture supernatant from the engineered strain into the optimized system. The supernatant was processed through the Amberlite IRC-50 column using the peristaltic pump, with waste fractions being systematically removed. Target ε-PLL was then eluted by applying 0.4 M acetic acid. For comprehensive purification, the eluate was subsequently passed through a configuration of two activated carbon columns. Finally, the purified ε-PLL's concentration was determined using ε-PLL content measurement method and the mass of the ε-PLL was determined using known concentration and compared against the crude supernatant's estimated ε-PLL mass to determine the productivity of the ε-PLL purification.
We also investigated the velocity of the liquid processed in the system. As shown in the video below, the speed of the liquid fueled by the peristaltic pump is significantly faster than that fueled by the air pump, enhancing the efficiency of filtration. In addition，carbon particles observed successfully filtered by the filtration cotton.
Learn
The productivity is relatively low, with about 2/3 lost, indicating that a step in this purification system is problematic. We talked to Dr. Zhu about this problem. She suggests that this is probably because of not enough binding time between ε-PLL in the bacterial culture supernatant and Amberlite IRC-50, recommanding us to elongate the binding time.
Cycle 3
A previous test indicated that the concentration of purified ε-PLL was lower than expected. Based on a recommendation of Dr. Zhu to increase the resin contact time, we tested the effect of elongating the running time on the Amberlite IRC-50 column.
Design
To increase the contact time between the supernatant and the resin, we modified the elution protocol by stopping the pump connected to the Amberlite IRC-50 column at beginning . This pause allowed the liquid to dwell within the column, promoting more thorough interaction. This process ensure the entire supernatant volume had passed through the column with extended contact time.
Build
Test
Comparison with the standard method (using continuous pump flow) showed that the mass of the eluted ε-PLL increased significantly. This confirms that without the pressure from the pump, the supernatant had more time to interact with the resin, allowing a greater amount of ε-PLL to be adorbed by the column.
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