Digestion of PET Plastic by Genetically Modified E. Coli in Simple Batch Style Bioreactors

Over the course of the 5 weeks, we ran a set of 5 batch-style bioreactors. They contained cultures of our genetically modified E. Coli suspended in liquid luria broth. We submerged plastic strips weighing roughly 0.6 grams into each of the bioreactors. Throughout each of the 4 weeks, we would meet 2-3 days to perform different tasks. These consisted of drying/weighing the plastic strips, cleaning the reactors, remaking the liquid cultures, and overall maintenance.

In the first week of the experiment we cultured 2 LB Agar plates with the E. Coli and the antibiotic ampicillin. It was made at a concentration of 100 µg/mL⁴. We used 2 plates to give ourselves a contingency plan in case 1 plate failed. Also, it allowed us to see how the 2 cultures affected our results. Over the course of the first week, the bacteria grew to the size as shown below. While it was easy to maintain a clean laboratory environment, it was difficult to maintain sterility. After a week of usage, the Agar plates became contaminated with a fungus. This forced us to replate the LB Agar plates weekly for the rest of the experiment.

In the second week of the experiment we created our first set of the bioreactors. For the 5 batch-style bioreactors, we created the following experimental setup. They consisted of one bioreactor which would be a control. This was placed in the incubator and had no bacteria present. It was labeled with a C. For the other four, the bioreactors were split into categories. For the first category, two bioreactors would be in incubator conditions and the other two would be in room temperature conditions. This gave them the label I or R. The second category was the LB Agar plate from which the bioreactor would receive their bacteria. Each plate was labeled with a 1 or a 2. These bioreactors were labeled as 1R, 1I, 2R, and 2I. We then cut out five 0.6 grams (± 200 milligrams) plastic strips from a PET plastic bottle. We measured all their weights on a Mettler scale which had a margin of error of 0.1 milligrams. After cleaning out the 125ml beakers used for the bioreactors with alcohol soap, we prepared 500ml of liquid luria broth. This was split equally among the 5 beakers. Finally, we introduced the bacteria from the plates into the bioreactors with an inoculating loop. After creating each bioreactor, we covered them with parafilm and poked a single hole through each bioreactor. This prevented contamination and allowed airflow into the bioreactors. Throughout this week, the bacteria grew as shown. Similarly to the LB Agar plates, the bioreactors became contaminated after a week. This forced us to create fresh bioreactors weekly. The plastic strips were cleaned and dried thoroughly for each new batch in our bioreactors.

During the 3rd week of our experiments we began weighing each plastic sample and cleaning out the bioreactors. Each plastic sample was removed from its respective bioreactor and then microwaved for a minute. After, it was patted down with a towel to remove residual moisture. They were individually weighed on a scale, and the measurements were recorded. After cleaning all the equipment with alcohol soap and disposing of the liquid culture, we remade each bioreactor and put them back in their growing environments.

During the 4th week of our experiments, we repeated the same process of weighing and drying each plastic sample. However, comparing the weights of the prior 2 weeks, we realized the increased porosity of the plastic samples would increase the water weight of them as the enzymes digested the plastic. To see if this created a significant enough extra variable, we performed a set of 3 different drying methods and weighed the plastic samples after each method. We first patted them down, microwaved them for a minute, and then we left the plastic samples in the incubator to dry for a day. Before we finished the day and remade the bioreactors, we conducted in-vitro staining on our sample of the E. Coli and the contaminant found in our LB Agar cultures. We confirmed the presence of our E. Coli, and an unknown fungus as shown below. We then remade each bioreactor, and put them back in their growing environments.

At the final week of testing there was a complication in our incubator. Both LB Agar plates we were using were dried out. Plate 1 was too compromised to recover a clean culture from, so we made a culture called 1’ from the bacterial stock. We were able to rehydrate plate 2 and made a new culture from that plate which we labeled 2’. For this week, we conducted the same tasks as we did during the first week of the experiment. Once we weighed all of the plastic strips, we were finished running the bioreactors. We terminated both cultures and cleaned out all of the equipment with alcohol soap.