The degradation of polyethylene terephthalate by Ideonella sakaiensis under varied

Conditions

Caroline L. Tsui, Dr. Todd D. Gruber Christopher Newport University, Department of Molecular Biology and Chemistry

Purpose of Project

The objective of this project is to investigate the degradation of polyethylene terephthalate (PET) plastic by Ideonella sakaiensis and discover the factors that can influence the rate of PET degradation. The bacteria Ideonella sakaiensis strain 201-F6 releases PETase an enzyme found to break down Polyethylene Terephthalate, or PET plastic.

Objective 1: Measure the rate I. sakaiensis degrades the mass of PET in perfect lab conditions

Objective 2: Determine if I. sakaiensis can eat powdered crystalline PET

Objective 3: Determine if there is a relationship between the hydroscopic attributes of PET and how well I. sakaiensis degrades the plastic.

Background Information

The most widely produced plastic in the world, PET is a polymer composed of ethylene glycol and terephthalic acid. These monomers are derived from raw petroleum and the plastic is prized for its durability and flexibility. But the same reasons that make it so useful to people also make it a huge hazard for the environment. Instead of decomposing, PET breaks down into tinier and tinier pieces called microplastics. These can accumulate and make their way up the food chain in a process known as biomagnification. And while PET plastic is recyclable, it degrades significantly during reheating. Every time it is recycled, the polymer chain grows shorter, and can only be recycled about 2 -3 times before its quality decreases to the point where it can no longer be used. A more common use for PET is downcycling, where lower grade products such as carpets or fabric are made. This form of recycling is known as open loop recycling, and is often seen as postponing disposal as these products are not recyclable.

The bacterium I. sakaiensis was discovered in 2016 in Sakai, Japan and can use PET as its sole carbon and energy source. It hydrolyzes PET by adhering to it and secreting the enzyme PETase, which effectively breaks down the PET into (mono(2-hydroxyethyl) terephthalic acid (MHET). I. sakaiensis then produces MHETase to break MHET down into the PET monomers of ethylene glycol and terephthalic acid. The discovery of an enzyme that can break down PET back into its monomers has huge implications for the future of plastic. If PETase could be engineered in a way to be involved in the recycling process, then there is a possibly of a closing the loop and allowing PET to be repeatedly recycled back into itself.

Research Objective 1: Rate of PET degradation

Determine the rate in which I. sakaiensis degrades a strip of PET plastic. Measurements of plastic weight will be taken every 24 hours.

Rationale: Once a solid function is created for the rate of PET degradation, other experiments can be compared to the original rate function to determine the effectiveness of that factor’s manipulation.

Steps

1. Place strips of PET plastic in 3mls of YSV media with inoculation of I. sakaiensis . Incubated in a shaker at 30 degrees.

2. Weighed every day at the same time.

Research Objective 3: Hygroscopic properties of PET

The amorphous PET that has been found to be edible by I. sakaiensis appears to have hygroscopic properties, meaning it is a material that absorbs water. This led us to question the relationship between hygroscopic plastic and degradation by I. sakaiensis

Rationale: Our laboratory has observed that the PET can grow in mass up to around .0009 g during the first three days before decreasing. This led us to questioning whether the extra weight came from the attachment of I. sakaiensis or whether the amorphous PET was absorbing fluid.

Steps

1. Strips of amorphous PET from blackberry containers or crystalline PET from water bottles were used for each trial

2. Blue contains distilled water, red contains YSV media, yellow contains media and inoculation

3. The PET was left in test tubes and weighed every day. On day three, after weighing, they were left out to dry.

4. They were weighed for two days until plastic returned to original mass

Expected Results

Research Objective 1: Rate of PET degradation by I. sakaiensis

The rate of PET degradation is difficult to capture because the PET gains the first couple days as it becomes waterlogged. As the bacterium multiplies, the mass is reduced accordingly. Results suggests that the plastic is reduced in mass by 0.0012 grams per day under ideal conditions. This can vary depending on how the different variables are altered.

Research Objective 2: Degradation of powdered crystalline PET

While I. sakaiensis cannot degrade crystalline plastic while in large strips, it was anticipated that a reduced surface area could possibly allow the bacteria to consume the crystalline PET.

Results show that I. sakaiensis is a hardy bacteria that can survive with no energy source for weeks at a time. Because powdered crystalline PET can not be weighed, another way to measure Ideonella growth will have to be discovered.

Research Objective 3: Hygroscopic properties of PET associated with degradation

Plastic weighed daily with a final equation of y = -0.0006x + 0.1474

Plastic weighed and 1 ml media replaced daily final equation of y = -0.0009x + 0.1349

Plastic weighted and centrifuged down with media replaced with a final equation of y = -0.0012x + 0.1497

Continued work: Replicate the data to ensure the equation is both precise and accurate

Research Objective 2: Degradation of powdered crystalline PET

Test to see if I. sakaiensis can use powdered crystalline PET as an energy source.

Rationale: I. sakaiensis only degrades amorphous PET, whose polymers are a random molecular jumble. It does not degrade crystalline PET where the molecular chains are locked in place against one another. Because water bottles are mostly composed of crystalline PET due to their stretch blow molding, it is important to see if I. sakaiensis will consume crystalline PET in any form. To test to see if I. sakaiensis can live on crystalline PET, we ran a trial where the surface area was reduced by powdering the PET.

Steps

1. One trial of nine test tubes all with 3ml of YSV media, the first three with a strip of amorphous PET, the second three with powdered crystalline PET, and the last three with nothing

2. After a week, a 20 μl inoculation was taken from each test tube and put in a test tube with 3ml and a strip of amorphous plastic.

Results: All inoculated tubes displayed growth, indicating that I. sakaiensis is a tough bacterium that can survive long periods of time without a food source. Because powdered crystalline PET cannot be weighted, an inoculation was used to determine if I. sakaiensis was alive. A different way to test growth will have to be explored.

Figure 3 displays that the amorphous PET plastic from the blackberry carton does indeed absorb liquid. Both the trials in the distilled water and the YSV media both increased in mass when left in liquid for three days, but went back to the original mass after sitting out and drying for two days. At the same time, the mass of the plastic in the YSV media and inoculation went up the first day and then begun trending downward because I. sakaiensis was degrading the plastic. Figure 4 displays the crystalline water bottle plastic does not absorb water and is also not degradable by the Ideonella

Continued work: Replicate this project by itself, but also with different types and forms of PET plastic (white PET from contact lens solution bottle) to determine if there is a relationship between hygroscopy and I. sakaiensis degradation.

To test the hygroscopic properties, PET plastic is immersed in water, media, and media with inoculation. Mass has been seen to increase for all on the first day, but the trial with media and inoculation was the only one seen to decrease.

Results suggest that the type of PET plastic that is hygroscopic is also degradable by I. sakaiensis . More trials with different forms of PET are needed.

Significance

Objective 1: General rates of plastic degradation are needed for comparison of different growth conditions.

Objective 2: I. sakaiensis exposed to powdered crystalline PET were alive after a week, but so were bacteria that had no plastic at all. Another experiment needs to be devised.

Objective 3: The observed increase in mass during experiments was explained due to the hygroscopic properties amorphous PET.

Acknowledgements

This work was supported by a 2018 Small Project Research Grant from VAS and by funds from Christopher Newport University to Professor Todd Gruber. Ideonella culturing procedures, and alternative ways to measure degradation were performed by Mary Adams, Andrew Chafin, Diamonte Jones, Carson Pittman (HRA), Sam Tyler, Noah Wallace, and Spencer West.

References

1. Austin, Harry P., et al. Proceedings of the National Academy of Sciences, vol. 115, no. 19, 2018, doi:10.1073/pnas.1718804115.

2. Bornscheuer, Uwe T. Science, vol. 351, no. 6278, 2016, pp. 1154–1155., doi:10.1126/science.aaf2853.

3. Tanasupawat, Somboon et al. International Journal of Systematic and Evolutionary Microbiology, vol. 66, no. 8, 2016, pp. 2813–2818., doi:10.1099/ijsem.0.001058.

4. Yoshida, Shosuke, et al. Science, vol. 351, no. 6278, 2016, pp. 1196–1199., doi:10.1126/science.aad6359.