STEM

Our STEM class focuses on reading and understanding technical writing, along with conducting our own independent research project which we present in February. I chose to focus my project on the issue of plastic pollution.

Use of PETase for PET Degradation in a Bioreactor to Improve Effectivity of Recycling

Overview

As industries continue to mass produce plastic, it is becoming more and more apparent that there is no effective way to recycle plastic and address plastic pollution. In fact, by 2050 it is expected that plastic production will grow by 70% to nearly 600 million tons per year (Cornwall, 2021). The majority of these products that are being made are made of non-biodegradable plastic polymers, and as the public do not take on sustainable disposal habits and societies fail to implement functional waste management systems, this plastic ends up polluting the earth.

The goal of creating a device that used bacteria to degrade plastic was not achieved at this time. For the first hypothesis that the mass would decrease, it was found that this was not supported since the p values were within .02 of 1. However, since the mass was taken in a way so as to not disrupt the biofilm, this was also measuring the mass of the bacteria, meaning that the increase in mass, although not significant, was likely the bacteria that were adapting to develop the enzyme to use the plastic as a carbon source. For the second hypothesis that the carbon dioxide would increase, this was also not significantly supported, with p values of 0.91 for all three groups. For the last hypothesis that the pH would decrease, this was not supported based on the p values of 0.78, 0.72, and 0.66 for PET, ABS, and PLA respectively.

Abstract

Bacteria with an adaptation to degrade plastic could aid in the recycling process in order for plastic to become less detrimental to the environment. As industries continue to mass produce plastic, it is becoming more and more apparent that there is no effective way to recycle it and address this pollution. The goal of this project is to engineer a device to effectively degrade plastic. Nine billion tons of non-biodegradable plastic waste pollutes the earth. While recycling is a good way to address this, manufacturers do not reuse plastic as freshly producing it costs less than recycled products. Some organisms have evolved from exposure to plastic and developed enzymes to break down plastic for energy. One type of plastic polymer is polyurethane terephthalate (PET). E. coli K12 was cultured in a liquid medium with PET film in a glass jar. The pH and carbon dioxide levels were recorded over a span of 168 hours at 30 degrees C. The mass of the PET film was recorded before and after and found to decrease as it was dissolved. Because CO₂ levels increased, cellular respiration was performed using carbon from the PET, which in turn made the solution more acidic. With this adaptation, these bacteria will be able to be used to dissolve plastic, and they can be cultured as needed to supply an industrial demand, overall creating a more effective system of recycling. In the future, isolating the products from this reaction could be investigated further to then use them to remake plastic, resulting in a cyclical process.

Graphical Abstract

Graphical Abstract

Research Proposal

Literature Review

Engineering Need

As industries continue to mass produce plastic, it is becoming more and more apparent that there is no effective way to recycle plastic and address plastic pollution.

Engineering Goal

The goal of this project is to engineer a device to effectively degrade plastic.

Background Infographic

Background

About 9 billion tons of plastic waste pollutes the earth. A majority of this was produced within the past two decades, evidence of the increased production of plastic in recent years to meet the higher demand due to its versatility. However, with this versatility comes a drawback, as these plastics do not easily decay in nature (Demircan & Keskin, 2020). Recycling is one process that aims to reutilize wastes that would otherwise add to environmental pollution. While recycling is a good way to address plastic pollution, most manufacturers choose not to reuse plastic as producing new plastics costs less than producing plastic from recycled products. Only 9% of all recyclable plastics end up being recycled in the US, since at every step of the process more and more of the recyclable material is disregarded, or it ends up in landfills and ultimately incorrectly disposed of (Sheth et al., 2019). Other ways to remove plastic waste is to burn it, but this releases pollutant gases such as sulfur dioxide, hydrogen cyanide, and hydrogen fluoride, which have been shown to be toxic and poisonous (Bellini, 2019). Burying it is another option, but this pollutes the land and water, and mechanically breaking it down is expensive (Demircan & Keskin, 2020). Alternatives to common uses of plastic, such as aluminum or glass cans, release more carbon into the environment than plastic, making them less beneficial alternatives (Stanton et al., 2020). Even when the material changes, humans will continue to litter, making it essential to find a way to actively recycle and properly dispose of wastes (Stanton et al., 2020).

Some organisms have evolved due to exposure to plastic and have developed enzymes to break down plastic for energy (Hiraga et al., 2019). Microorganisms found in areas containing trash and vegetation, namely plastic landfills, have adapted to contain enzymes that can decompose plastic polymers, including the polymers that are used in bottles and clothing (Cornwall, 2021). One organism, Ideonella sakaiensis 201-F6, was found in a landfill and was taken to lab and found to have properties to degrade PET. These properties were enzymes, namely PETase, which can dissolve PET by using it as a source of energy and carbon and ultimately breaking PET down into its monomers (Hiraga et al., 2019). Ester bonds link PET monomers which are hydrolyzed – or broken down with water - by hydrolase enzymes in nature, allowing the plastic to be biodegraded by breaking the carbon-carbon bonds (Hiraga et al., 2019). These carbon-carbon bonds are what make the polymer more difficult to degrade when compared to other plastics such as polyesters that have carbon-oxygen bonds (Cornwall, 2021). The bacteria latch on to the PET and form a biofilm - a colony of bacteria that rely on the material for nutrients (O'Toole et al., 2000). However, some enzymes have been deemed to not work fast enough and others are only able to break down a specific plastic polymer, and these qualities are undesirable to the general public as it is harder to implement this technology when it takes a long time for the process to be completed (Cornwall, 2021). These limitations have prompted studies to improve the enzyme. While many experiments have looked at ways to improve the efficiency of PETase, little progress has been made to utilize this technology at its current functionality to address the growing issue of plastic pollution and ineffective recycling, and little research has been done to try and culture bacteria with this enzyme without expose from a landfill or what conditions may be needed for this to be possible.

Procedure Infographic

Procedure

The procedure by Odobašić was followed to make the YSV media and culture the bacteria (2020). 200 mL of the media was placed in a mason jar, and plastic filament was sterilized with ethanol and placed in the jar with a stir bar. An incubator was used to culture the bacteria, E. coli K12, for the duration of the experiment, and the stir plate was place in the incubator with the four jars on it and taped shut.

A pH meter was sterilized with bleach after each use in order to measure the pH of the solution to see if the carbon from the plastic filament was being used. A CO₂ sensor was used by placing it through a hole in the top film in order to observe if the carbon from the plastic filament was being used. Once it reached a steady reading, this number was recorded, and the jar was recovered and placed back in the incubator. The mass of the plastic was measured before and after the experiment occurred so as to not disturb the formation of a biofilm on the plastic in order to measure if the plastic had been significantly degraded

Carbon dioxide chart pH values graph

Analysis

Changes were observed in regard to the pH, carbon dioxide levels, and mass of the different filament groups in this experiment.

It was found that the pH in all groups went up and then began to decrease. The pH of PET was 8.31 112 hours in, and at 168 hours it had decreased to 7.55, however, a similar trend was seen in all groups as detailed in Figure 1. From the Chi Square tests, p values of 0.782, 0.718, and 0.655 were found respectively, signifying that there was not a significant change from the base pH in either group.

It was found that the carbon dioxide levels increased for PET and PLA filament groups by an average of 6 ppm, but not for the ABS filament group over the span of 168 hours as seen in Figure 2. Based on the p values of 0.91 from the Chi Square tests, there was not a significant change in carbon dioxide levels over the span of the experiment.

When comparing the masses before and after the testing, the mass for PET increased by 0.02 grams, while the mass for PLA increased by 0.0075g and the mass for ABS decreased by 0.0167g. Based on the Chi Square test, these changes in mass were not significant, resulting in p-values of 0.983, 0.985, and 0.994 respectively from the Chi Square tests.

Discussion

The goal of creating a device that used bacteria to degrade plastic was not achieved at this time. For the first hypothesis that the mass would decrease, it was found that this was not supported since the p values were within .02 of 1. However, since the mass was taken in a way so as to not disrupt the biofilm, this was also measuring the mass of the bacteria, meaning that the increase in mass, although not significant, was likely the bacteria that were adapting to develop the enzyme to use the plastic as a carbon source. For the second hypothesis that the carbon dioxide would increase, this was also not significantly supported, with p values of 0.91 for all three groups. For the last hypothesis that the pH would decrease, this was not supported based on the p values of 0.78, 0.72, and 0.66 for PET, ABS, and PLA respectively.

One limitation was the accuracy of data due to the testing environment. Since the testing location was in a classroom, the carbon dioxide level in the room was higher at the end of the day by about 200ppm, skewing the readings of the jars since they were not set up in a completely closed environment. To address this, carbon dioxide levels were analyzed as the difference between the room level and the observed reading. Additionally, because the testing period was limited, the recorded results were not as extensive as expected.

Chi Square tests were used to compare the initial results to the final results after a period of time to see if there was any statistically significant change in the readings. It was found that there were no significant differences between the initial and final readings for the mass, carbon dioxide, and pH measurements. While many past studies looked at culturing Ideonella sakaiensis, this experiment attempted to culture bacteria that could carry out the same process as I. sakaiensis. Because of this, there would be a greater ability to culture these bacteria as needed to supply the industry with a device to degrade plastic.

In the future, more research could be done to optimize the media for the growth of plastic-degrading bacteria. This would allow for more ideal growth of bacteria, and the creation of a cyclic process for recycling so that less plastic ends up in landfills and polluting the earth. Additionally, the next step would be to develop a process to separate the ethylene glycol and terephthalic acid from the rest of the solution and media to be able to reuse these substances for plastic.

References