Team:Westminster UK/Naturaldegradation
Natural Degradation Experiment
Introduction
Aside from disposed polystyrene waste of which about 10% gets recycled, massive amounts of polystyrene waste end up being thrown away inappropriately, resulting in natural contamination. A previous iGEM team from the University of Leicester focused on testing the end life cycle of polystyrene and investigated the presence of polystyrene degrading bacteria in below-surface conditions through their citizen science experiment. The results of their experiment demonstrated low reduction of polystyrene volume, while identifying two strains of bacteria capable of growing in conditions adhering to polystyrene. Although their data currently offers an insight to polystyrene degradation on land, massive amounts of eroded expanded polystyrene (EPS) also end up in rivers and eventually accumulating in the ocean. In early May 2018, a microbiology team from the University of Westminster went on a field trip towards north-east England, on the way collecting river and mud samples that they kindly offered to our team for testing. In outside conditions, polystyrene plastics are also exposed to ultraviolet (UV) radiation, which has additionally been found to have an effect on polystyrene degradation (Yousif, 2013). The aim of this investigation was to detect the presence of polystyrene degrading microorganisms in the given river samples by detecting changes in the mass of polystyrene when exposed to river water and mud conditions.
Method
Out of 8 sample locations that were provided for analysis, four of the most geographically dispersed sets of river and lake samples were collected, being mainly from different regions of east England (See figure 1). The samples used were selected from the following locations:
1. River Nar
2. Lake Thompson
3. Alconbury Brook River
4. River Tove
To create the control, samples from the four locations were mixed in a 10 mL tube and autoclaved, to ensure no living microorganisms are present.
For each location, one mud sample and two water samples (with UV and without UV conditions) were exposed to two types of polystyrene for 3 months at an average temperature of 19 °C. The polystyrene samples used include expanded polystyrene (EPS) in the form of beads (covered in fire retardant) and a dense, non expanded form of high-density polystyrene (HDPS) in the form of strips, cut out from cups collected from the University of Westminster Faculty of Science and Technology cafeteria.
EPS beads were purchased from eBay:
HDPS cups were purchased from:
https://johndarvellpackaging.co.uk/PS260W-Box-of-Disposable-Polystyrene-Cups-260-ml-92-floz
The HDPS cups were divided into 0.2 g of 2 cm x 0.5 cm strips and 0.2 g of the EPS beads were measured by placing beads one by one on the precision scale to get to the desired mass above 0.2g. In order to reduce the inaccuracy of measurements, each polystyrene sample was weighed three times before and after the experiment. The samples were placed in glass test tubes, and each sample contained 5 mL of river water and mud. They were placed in tube racks, which were placed in a fume cupboard to maintain constant temperature and to eliminate any unpleasant scents.
The non-UV treated samples were covered in loosely sealed aluminium foil and small holes were cut to allow oxygen flow.
All samples were regularly monitored at weekly intervals.
UV conditions were performed as followed:
1. The samples were placed under a long wave UV lamp, with the whole system covered with an aluminium foil in order to increase the exposure of the radiation towards the samples.
2. The lamp’s on/off cycle was regulated by a timer wall plug, which was set to periodically switch the radiation exposure every 12 hours.
3. The samples were prepared and set to exposure on 29th of June 2018.
Figure 1: The mass of the polystyrene (EPS & HDPS) before the experiment
Table 1: The natural degradation experiments set up in a fume hood. The two images provide a rough outline of the final sample distribution: The left tube rack contains EPS samples mixed in river water and mud. Additional controls were made both for water and mud samples, located in the last row (two of each). The right picture was taken from below, looking directly at the 10 UV treated samples.
Results
The results were obtained on the 29th of September 2018, exactly three months after initial exposure. Visually, the non-UV treated samples did not display any significant changes, whereas in the UV treated samples, the river water had evaporated. Once obtained, tubes with river water samples were decanted, the polystyrene beads were then washed off with distilled water, blot dried and measured on a precision balance. Each sample was triple measured and an average was taken from their values. The results from the obtained samples gave higher values. It was assumed that this was due to their recent collection, so they were left to dry for another 24 hours. The next day the samples were weighed out and the weight changes were calculated and recorded:
Figure 2: Mass changes of EPS and HDPS in river water samples.
The river samples with mud required more preparation: As the polystyrene samples were dug within the mud, taking them out required extensive test tube washing and mud cleansing from the surface of the plastics. The beads were rubbed on average for a minute each. After washing, the samples were kept to dry for 24 hours. The weight changes were calculated and recorded:
Figure 3: Mass changes of EPS and HDPS in mud samples.
Discussion
One major observation with all the results is that the apparent mass change recorded in all the samples ended up being negative, meaning that all the samples lost a portion of their weight. However, a notable observation can also be seen in the control samples, indicating that even if microorganisms were present, no data correlation could prove their biodegrading abilities. To explain why the control samples also demonstrated a change in mass, factors that have occurred during the cleaning and measuring procedures should be included. For example, the EPS samples may have lost mass from scraping during the cleaning procedure, which can be proved through a few comparisons:
-Comparing EPS mass loss to the mass loss of the high density polystyrene samples: The high density polystyrene samples are harder, and thus less prone to being eroded during the cleaning procedures. When observing the results, this can be confirmed, as the hard density samples did demonstrate a lower loss of mass.
-Comparing EPS mass loss in non-UV water samples to mud samples. Because the water samples did not go through the rubbing procedure as the mud ones did, they may have retained more mass during the cleaning procedure. When observing the results, this can be confirmed, as the mud samples did demonstrate a greater loss of mass.
On the other hand, a correlation can be found in the UV treated samples when comparing them to the non-UV treated ones. The change is stronger in the EPS samples, which can be explained by their low density that may allow more UV radiation to pass through and allow it to be more broken down.
Conclusion - Does polystyrene break down in river waters?
Polystyrene waste in natural waters poses a threat to the biosphere as ingested polystyrene plastics may cause gastrointestinal blockages or even introduce toxic and carcinogenic pollutants into them. In this experiment, we wanted to see whether certain conditions in rivers and lakes of england contain microorganisms that may facilitate the natural degradation of polystyrene. Apart from biodegradation, UV radiation was included as a natural element of exposure to polystyrene. The obtained data was not sufficient to prove biodegradation by microorganisms, partially due to inaccuracy of the results, and partially due to other procedures that could have been (and were not) done to detect bacterial presence. UV radiation was found to break down polystyrene with x4 efficiency for EPS and x3 the efficiency for HDPS (biased results).
To improve the biodegradation side to these types of experiments, polystyrene samples could be surface-scraped and sequenced for present bacteria through 16s sequencing. Further research could then be done by other scientists by examining the sequenced genome through bioinformatic analysis.
References:
Yousif, E., Haddad, R. (2013). Photodegradation and photostabilization of polymers, especially polystyrene: review. Springerplus. 2: 398.
