Hardware

In order to effectively decompose cellulose of pine tree, we created a “Reaction Machine” that can react with pine tree powder and 4 kinds of enzymes were manufactured. The reaction conditions were set at 37°C and pH 5.0, and the impeller was designed to operate at 20 rpm in an environment where 60-70% humidity was maintained.

1. Reaction Machine

In order to make the pine tree powder and enzymes react, we modified the shaking incubator (C-SKI-2, Changshin Science) and performed modeling using the Autocad program. We installed the reaction chamber (270x330x300 mm) inside the incubator and made a space by separating the existing shaking part. The “temperature control system” and timer of the existing “shaking incubator” can be used as it is.

2. Impeller(Blade)

A plastic reaction chamber (270x330x300 mm) was installed inside the shaking incubator, and 4 impellers (blades) were connected with the shaft in the center of the chamber. Also, a chain was installed on the side of the chamber and connected to the motor to rotate the impeller. The impeller rotation speed was set to be maintained at 20 rpm.

3. Arduino

To decompose the cellulose of the pine tree powder, 4 types of enzymes should be added, and citrate buffer (pH5.0) should be supplied to increase the activity of the enzymes. For this purpose, a 12V DC motor was installed through Arduino (Uno) control. As a result of measuring the activity of cellulase cocktail (KsCel7A, Tfcel6B, LFCA_EG, and CtBglA) by time in the previous experiment, the activity of the cellulase disappeared after 24 hours. By applying this to hardware, it was programmed to inject 50-100mL of cellulase cocktail per hour based on 10L of pine tree powder. The cellulose cocktail was prepared by overexpressing a total of 4 celluase 2 types each in B. subtilis and E. coli.

4. Sensor & Pump

The temperature of the incubator was set at 37°C using a shaking incubator, and a temperature sensor was additionally installed to check the temperature more accurately. In addition, the humidity sensor was installed so that the humidity inside the incubator could be maintained at 60~70%. Humidity was measured through a humidity sensor, and a certain amount of 50 mM citrate buffer (pH 5.0) was injected based on the data value. Since it was impossible to measure the powder-type sample using a pH sensor, a sample was obtained from the reaction chamber every day to measure the pH, and this was reflected in the data value.

5. UV Lamp & Combustion Tube

As shown in the figure above, an ultraviolet sterilization lamp (30W, Philips), a metal catalyst combustion device (N Biotech Co., Ltd.), and a HEPA filter were installed in succession. The incubator is not a completely enclosed space, and bacteria may leak out during the reaction process. A vacuum pump was installed at the bottom of the incubator to prevent leakage. The internal air is continuously passed through a UV lamp and a metal catalyst combustion device to sterilize the exhausted air, and oxides generated in this process are removed by a HEPA filter.

6. Conclusion

The reaction machine made by remodeling the existing shaking incubator worked better than we expected. There were team members who did Robotics-related activities, so it was possible to manufacture without much help from outside, and it was possible to save production costs by using recycled mechanical parts and Arduino. It took about two months to design and manufacture, and it has been in operation for more than 11 months so far. The inconvenience during the experiment was that the tube to which the cell media was connected had to be replaced every day to prevent contamination, and there was also the inconvenience of supplying cultured bacteria every day. Therefore, we are designing an automated system that starts with bacterial culture, induction, and supplies cultured bacteria directly to the Reaction Machine. If this automated device is completed, it will be possible to obtain a large amount of Pinesorb more conveniently, and we think it will be helpful for researchers who need similar experiments and processes.