June 2016 Volume 3, Issue 4

Determining the Potential Secondary Impacts Associated with Microorganismal Biodegradation of Microplastics in the Marine Environment

Mehr Kumar1* #, April Xie1*, and Jackie Curley2
Student, Teacher2: The Academy of Science, 21326 Augusta Drive, Sterling VA 20164
*These authors contributed equally
#Corresponding author: plastisphereusa@gmail.com

Abstract

Microplastics account for 80% of the 8 million tons of plastic pollution introduced to the oceans annually. Persistent organic pollutants (POPs), chemicals found in low concentrations in the oceans, do not degrade easily. POPs concentrate on hydrophobic substances like microplastics and bioaccumulate. Research indicates that marine microorganisms can degrade microplastics in the marine environment. This study explores the environmental impacts associated with the biodegradation of plastic by studying degradation products of biodegraded polyethylene terephthalate (PET) microplastics by microorganisms in the marine environment. The trial water and contents of the lysed cytoplasm of two marine microorganisms, one species of bacteria and one fungus, following biodegradation of the microplastics were tested using Fourier Transform Infrared Spectroscopy (FTIR) to separate and identify remaining substances. FTIR spectra comparisons of experimental and control trials for both types of trials showed negligible differences. This implies no detected difference between byproducts in control and experimental trials. Thus, there has been no evidence to suggest that the biodegradation process released plastic byproducts into the surrounding marine environment or into the cytoplasm of the microorganisms.

Introduction

Plastics, comprised of large organic molecules, are synthetic polymers that originate from materials extracted from oil, natural gas, and coal. They contain long carbon chains as backbones made of repeated units created during the polymerization process1. About 245 million tons of plastic are produced each year and around 30% of all plastic products are created for packaging purposes2. Polyethylene accounts for almost 64% of disposable plastics3. Polyethylene terephthalate (PET or PETE) is a semi-crystalline aromatic polyester polymer that is especially durable, and therefore a popular material for manufacturing containers and other packaging, most notably plastic drinking bottles4,1.

Due to their durability and buoyancy, plastics have not only the ability to move and disperse throughout the oceans, but are resistant to biodegradation, causing them to remain in the environment, and especially the marine environment, for long periods of time. In fact, most plastics are deemed not biodegradable. Synthetic plastics are not easily biodegraded in the natural environment due to their hydrophobicity and decreased solubility5. PET would ordinarily be easy to break down, but aromatic polymers, giving the plastic qualities such as heat-resistance, make it much more resistant to degradation. Due to this, PET is almost non-biodegradable under normal conditions6.

Microplastics, of particular concern, are particles up to 5 mm in size as indicated by the National Oceanic and Atmospheric Administration (NOAA)7. They account for the majority of marine plastic pollution8, over 80% of plastic debris on the ocean floor9. These microplastics have been found globally in the oceans, even in Antarctica8.

Since PET is almost non biodegradable under normal conditions6, any biodegradation involves two steps: the exposure to an environmental factor that alters the chemical makeup by reducing the molecular weight, followed by the degradation by microorganisms10. Under exposure to UV-radiation, plastics often undergo photo-oxidation which allow the plastics to be more easily metabolized by microorganisms11. Previous research indicated that UV radiation of microplastics facilitates biodegradation12.

Persistent organic pollutants (POPs) are synthetic materials that are found in low concentrations throughout the oceans. They are stable, hydrophobic, and lipophilic chemicals that do not degrade easily. POPs also bioaccumulate, and are carried up the food chain as they stick to fatty tissue13.

As these toxins accumulate on microplastics, it becomes much more likely that these toxins will be introduced into the food chain through the ingestion of these smaller plastic fragments by marine organisms2. A study explored the toxicity of POPs in the context of marine microplastics13. This study found that microplastics do, in fact, exist as a vehicle and harbor of these toxins. In addition, microplastics have greater surface area than macroplastics do, making them even more susceptible to carrying POPs.

This indicates that that the microplastics themselves are not of considerable danger to marine organisms, it is toxins they carry. While macroplastics may also gather these pollutants, due to their size, they are not nearly as dangerous as microplastics because they are not as easily ingested by marine organisms.

The problems in the field of biodegradation of microplastics or polyethylene terephthalate is mainly due to the high resistance of the plastics to biodegradation. The hydrophobicity, high molecular weight, and minimal solubility all assist in deterring microorganisms from degrading polyethylene. Microorganisms also tend to not degrade polyethylene if they have another source of carbon available, therefore creating a situation in which the plastics must be the only carbon source for the microbes, or they will not degrade them at all.

Previous research by these authors12 demonstrated that biodegradation of microorganisms Shewanella purtrefaciens and Streptomyces viridosporus,  found in the marine environment, have the ability to degrade PET microplastics. So far, not much research has been done on other aspects of microbial biodegradation such as the mechanisms of biodegradation, the specific enzymes involved in the biodegradation process, and the fate of the plastics after they are consumed by microorganisms14.

While the biodegradation of plastics has been studied in the marine environment, the state and location of the biodegradation products following this process has not. Determining what products, including potential toxins, remain provides an avenue to evaluate the true efficiency of this process; if the process yields more toxins as a result, biodegradation may not be a safe solution to marine plastic pollution.

The purpose of this study was to analyze the environmental impacts of using marine microorganisms as a means of biodegrading microplastics in the marine environment by identifying any potential secondary byproducts associated with the process. It was hypothesized that biodegradation of microplastics will not release byproducts into the surrounding marine environment. It was also hypothesized that biodegradation of microplastics will not introduce byproducts into the cytoplasm of degrading microorganisms.

Materials and Methods

PET water bottles (post-consumer use) were cut into smaller pieces, about 2 square inches or smaller. The plastics shredded using a kitchen blender for 20 minutes with water. The plastics were poured through a 5 mm sieve to verify the size of the plastics to fit the definition of microplastics. A 35 ppm saltwater solution was created using Instant Ocean© and deionized water. The microplastics were exposed to a UV-A lamp for 96 hours, swirling continuously in saltwater solution.

Shewanella putrefaciens  (ATCC Catalog Number 8071) and Aureobasidium pullulans (ATCC Catalog Number 9348), all with a biosafety level 1, were rehydrated and maintained following the ATCC protocols at 26°C.

Experimental trials were prepared by adding 75 mL of  saltwater solution to a flask. 0.5 grams of dry UV-treated microplastics were measured and added. In a sterilized environment (LFC 10562-008), 0.50 AU of the first organism was added to each flask. They were all sealed with aluminum foil and left to incubate for two weeks. Using a coffee filter, the solution was strained to separate plastics, which were vortexed in sodium dodecyl sulfate to remove biofilms and left to dry, and the remaining solution was kept in centrifuge tubes for further analysis. The final dry mass of the plastics was measured to ensure that degradation had taken place.  All previous steps were repeated with each species.

Two controls were run alongside experimental trials for comparative analysis. The first involved 75 mL of saltwater solution in a flask, the second involved 75 mL of saltwater solution in a flask with 0.5 grams of UV-treated microplastics.

To prepare the remaining solutions following biodegradation of microplastics in the isolated marine environment periods for analysis, the remaining water was kept for two purposes: to identify substances remaining in the water and to identify substances in the cytoplasm of the cells. For each trial with saved water, 0.5 mL of water was mixed in a microcentrifuge tube with 0.5 mL of hexane. Additionally, a culture was grown up from the remaining saved water. After 48 hours, these organisms were lysed to expose the cytoplasm of the cells. Once again, 0.5 mL of this lysed solution was mixed with 0.5 mL of hexane. Once the hexane settled on the top due to a lower density, the solution on the bottom was discarded. A control with the buffers used to lyse the microorganisms and mixed with hexane in the same way to create a comparison for later analysis. The solutions for each trial, one analyzing the lysed microorganisms and the other analyzing the contents of the water, were tested using Fourier Transform Infrared Spectroscopy by obtaining spectra for each sample for later comparison. FTIR spectra were used to identify the constituents of each sample and were compared among relevant controls. These steps were repeated for each sample from all trials. An explanation of trials and the relevant controls used for FTIR are listed in Table 1.

 

Results

In order to determine the byproducts that remained in the marine environment following degradation, the FTIR spectra (Figure 3), which shows the experimental trials testing the marine environment (Trials 1, 2, 3a, 4a, 5a), were compared to two controls: the water control (Figure 1) and the plastic control (Figure 2).

 

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Figure 1. Water Control Spectra.

Figure 2. Water & Plastic Control Spectra.

Figure 3. Water Control, Water & Plastic Control, and Water Experimental Spectra. Green spectra is the water control, blue spectra is the water and plastic control, red spectra is the water experimental.

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    Figure 1. Water Control Spectra.
  • fig2
    Figure 2. Water & Plastic Control Spectra.
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    Figure 3. Water Control, Water & Plastic Control, and Water Experimental Spectra. Green spectra is the water control, blue spectra is the water and plastic control, red spectra is the water experimental.

 

In order to determine the byproducts that remained in the cytoplasm of the cells following biodegradation, the FTIR spectra (Figure 5), which shows the experimental trials testing the cytoplasm of the cells (3b, 4b, 5b), were compared to three controls: the water control (Figure 1), the plastic control (Figure 2), and the cell lysis control (Figure 4).

In both cases, the experimental spectra were consistent with the control spectra (Figure 6). Additionally, the spectra for the experimental trials that tested the marine environment (Trials 1, 2, 3a, 4a, 5a), the experimental trials testing the cytoplasm of the cells (3b, 4b, 5b), and all three controls (Figures 1,2,3) all matched one another. The polyethylene terephthalate graph (Figure 7) shows the FTIR spectra for a PET microplastic in the context of the control and experimental spectra in Figure 6. The two experimental FTIR spectra were not consistent with the PET spectra.

 

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Figure 4. Cell Lysis Control Spectra.

Figure 5. Cell Lysis Control, Cell Cytoplasm Experimental. Green spectra is cell lysis control, red spectra is cell lysis experimental.

Figure 6. Controls and Experimental Comparison. Purple spectra is water control, blue spectra is water experimental, green is water and plastic control, red is lysed control, orange is lysed experimental.

Figure 7. Polyethylene Terephthalate Comparison. Light blue spectra is PET.

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    Figure 4. Cell Lysis Control Spectra.
  • fig5
    Figure 5. Cell Lysis Control, Cell Cytoplasm Experimental.Green spectra is cell lysis control, red spectra is cell cytoplasm experimental.
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    Figure 6. Controls and Experimental Comparison. Purple spectra is water control, blue spectra is water experimental, green is water and plastic control, red is lysed control, orange is lysed experimental.
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    Figure 7. Polyethylene Terephthalate Comparison.Light blue spectra is PET.

Discussion

Some marine microorganisms have the ability to biodegrade microplastics in the marine environment. However, the degradation process and enzymes involved are currently not known. This study investigated the potential byproducts released into the surrounding environment or into the cytoplasm of the degrading cells following biodegradation of microplastics. It was hypothesized that no byproducts would be found in either of the two investigated areas, the surrounding marine environment and the cytoplasm of the degrading cells, since PET plastic is not petroleum based and can potentially break down completely into CO2 and water.

The fact that there was negligible difference between the experimental and control spectra implies that there is no detected chemical difference between these trials. Therefore, these results also imply that there was no evidence that biodegradation process released plastic byproducts into the surrounding marine environment or into the cytoplasm of the degrading microorganisms.

Based on the consistency of the FTIR spectra (Figure 6), no biodegradation intermediates were detected. This evidence points to possible complete biodegradation of the PET microplastics. PET has a chemical formula of C10H8O4 (Figure 7). Other types of polyethylene have been shown to degrade into CO2 and H2O as a result of biodegradation pathways in which the polymer is metabolized after entering the citric acid cycle. This evidence suggests this may also be the case for PET, making its biodegradation safe. These results support our hypotheses that the biodegradation process of PET microplastics will not release byproducts into the marine environment or the cytoplasm of the degrading microorganisms.

The polyethylene terephthalate graph (Figure 6) shows the FTIR spectra for a PET microplastic. The two experimental FTIR spectra (Figure 6) were not consistent with the PET spectra. This indicates that the biodegradation process did not simply create even smaller PET microplastics.

Finally, all spectra showed evidence of two specific peaks: one identified as CO2, and the other as polyethylene. The presence of CO2 can be attributed to microorganismal respiration and biodegradation products. The evidence of polyethylene in all control and experimental trials may be due to the fact that much of the substances utilized in this procedure were stored in a plastic container at some point.

The reduction of microplastic pollution translates to the reduction of marine organism deaths caused by plastic pollution and, more importantly, deaths caused by the toxins they carry. Determining the environmental impacts associated with using biodegradation as a method of combating marine microplastic pollution aids in distinguishing safe methods for solving the marine plastic problem. Further research should test the ability for each species to degrade various POPs and replicating this process on microplastics from the ocean.

Despite the consistency of the results across more than 80 trials, there are a number of factors that contribute to limitations of these results. First, it was assumed that when samples were isolated from the cell lysis buffer solutions, each sample contained the contents of the cytoplasm of the lysed microorganisms. Thus, it was assumed that each of these samples, when tested using FTIR, was representative of the contents of the cytoplasm of the lysed cells following biodegradation of microplastics. Additionally, it is possible that the use of hexane as a nonpolar solvent did not attract the substances in the water, and thus, the presence of those substances were not accurately tested using FTIR. It is also possible that the zinc crystal on the FTIR was not properly cleaned between trials, confounding the results. Finally, the FTIR libraries that were used to compare the spectra with and identify substances only contained the spectra of substances already run through FTIR or from standard spectra gathered from other sources. Therefore, the specific substances released into the surrounding environment or into the cytoplasm of the microorganisms following biodegradation could possibly have not been stored as a comparable spectra in the libraries used in the analysis. If this was the case, then the by products of biodegradation would have never been detected in the first place.

Thus far, there has been no evidence to suggest that biodegradation of microplastics releases byproducts into the surrounding marine environment, supporting our first hypothesis. Additionally, there has been no evidence to suggest that biodegradation of microplastics introduces byproducts into the cytoplasm of the biodegrading microorganisms, supporting our second hypothesis.

The reduction of microplastic pollution translates to the reduction of marine organism deaths caused by plastic pollution and, more importantly, deaths caused by the toxins they carry. Determining the environmental impacts associated with using biodegradation as a method of combating marine microplastic pollution aids in distinguishing safe methods for solving the marine plastic problem.

Future work in this field should focus on determining what remains on the plastics following their removal from the water, testing the ability for various species to degrade POPs, and replicating this process on microplastics from the ocean.

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Acknowledgements

We thank Dr. Michael Tomlinson for FTIR assistance and advice.