Hanna Kessler
Boise, ID
2016, Senior, Creative Writing

Abstract

Discarded plastics enter the ocean, causing problems for marine organisms and humans. Current solutions ­­ recycling, burning, and biodegradable plastics ­­ are not effective enough to curb increasing production. Pseudomonas, a freshwater bacteria, can degrade low density polythene (LDPE) and polyethylene terephthalate(PET) plastics using PHA synthase, which converts plastics into bioavailable polyhydroxyalkanoate (PHA). Plasmids containing the gene for PHA synthase, controlled by a ligand, along with an antibiotic resistance (ampicillin) will be incorporated into a marine bacterium, Pseudoalteromonas sp. D41. Artificial selective pressure in the form of ampicillin will be used to insure incorporation of the plasmid. The efficacy of Pseudoalteromonas sp. D41 at degrading plastics will be tested in vitro. It will be incubated in falcon tubes along with LDPE plastic and the ligand, with the mass loss compared to the results of a previous study on Pseudomonas, along with a set of sterile control tubes, a set containing unaltered Pseudoalteromonas sp. D41, and a set without available ligand. Expected mass loss is between 7 and 17 percent in 40 days in tubes with both ligand and altered Pseudoalteromonas sp. D41, based on the study, with additional mass lost at 60 days. Additionally, cultures will be taken to test the amount of bacteria present. Bacteria is expected to be present in all of the tubes except the control. Presence of bacteria in the control tube indicates contamination, and nullifies the results.

Introduction

Description of Problem
In the modern era, plastics are a common and well known material. 2013 alone saw 299 million tons of plastic produced worldwide. This was a 4% increase from the prior year, which follows the pattern of steady increase year over year. They are cheap, easy to produce, and are non­biodegradable. All of these traits make them a great material for such applications as packaging, plumbing, car parts, and toys.

However, these properties also make them a growing and persistent problem. As much as 12.7 million tons of plastic end up in the ocean each year. This plastic comes from many sources. It is blown off the backs of garbage trucks and out of landfills, and is then carried down rivers and sewers until it eventually reaches the ocean. The aquaculture industry is also a major contributor with plastic floats, fishing lines, nets and packing bands, which are often cut free and abandoned (The Problem, n.d.). Once the plastic is in the ocean it accumulates in gyres, which are large ocean current systems. Gyres are caused by the Corvallis effect and strong winds along with the ocean currents. Plastic will form large floating clumps in the places where these currents converge. Here a bag can last 20 years, and a bottle can last upwards of 450 years. There are gyres in most of the major oceans, and all of them contain plastic debris. The largest of these are the Western Pacific Garbage Patch ­­ located between Japan and Hawaii ­­ and Eastern Pacific Garbage Patch ­­ located between Hawaii and California. They are often collectively referred to as the Great Pacific Garbage Patch, and have a combined area greater than twice the size of Texas. The amount of plastic in them is staggering, and only increasing by the day.

Impact
Marine life is affected by the plastic debris in our oceans. Floating plastics block light from reaching lower parts of the ocean, which can stop important phytoplankton. These are the primary producers for much of the open ocean, so damage to them affects the entire ecosystem. Pieces of plastic can sink and smother marine plants. Floating islands of plastic create an artificial habitat for animals, bacteria and plankton. As the flotsam moves, it carries these organisms with it, potentially introducing invasive species. Many marine animals get tangled in nets and other plastic products and drown, starve, or become injured. Some animals mistake the plastic for food and consume it. Small bits of plastic can commonly choke birds to death or clog their digestive systems. Toxic materials in the plastic can cause abnormalities in the liver and stomachs of fish.

Additionally, these toxins and undigested plastic pieces can be passed up the food chain, building up higher concentrations in predators such as tuna and salmon. Not only can this kill these key predators, potentially throwing ecosystems off balance, but dangerous and toxic chemicals can be passed onto humans who eat them.

There are several toxins found in plastics that are considered dangerous to humans. These include bisphenol A, which can cause insulin resistance leading to heart disease and is linked to cancer; polyvinylchloride, which has been known to cause cancer, birth defects, and chronic bronchitis; and phthalates, which may cause infertility, immune system impairment, and cancer.

There are other negative effects to humans caused by the amount of plastic in the ocean. Litter and plastic in and surrounding oceans decrease the popularity of coastal locations and affect tourism rates, and thus the local economy. For example, Kamilo Beach used to be one of the most beautiful places in Hawaii, but it has become one of the most polluted. Garbage that is washed up from the Great Pacific Garbage Patch covers the beach; up to 90% of it is plastic. Before local residents started cleaning the area, the piles of garbage were as high as ten feet tall in some places. Money must be spent cleaning beaches. Approximately $13 billion is spent a year is attributed to these costs, yet the problem is still growing.

Current Solutions
Often a lack of education leads the public to believe that plastic problems are mostly resolved by the current solutions, but this simply isn’t true. While efforts are being made to recycle more, still only 9% of plastic in the United States are recycled. The rest is put into landfills, where lightweight plastic is easily blown into rivers and streams, eventually ending up in the ocean. In Europe, some plastic is burned as an alternative fuel source, which requires large, expensive equipment to prevent releasing toxins into the air. Even with these measures only about a third of the plastics used there are destroyed. Large amounts of plastic are sent over to developing countries ­­ such as China ­­ where they are often processed in small scale facilities without proper care or equipment, and further damage the environment.

Biodegradable plastics offer a partial solution, however they are not appropriate for many of the most common uses of plastic. Because they are not bacteria resistant, they aren’t usable in food packaging or in applications that need to last a long time. This makes them problematic as a replacement to conventional plastics.

Even if no more plastics were produced, the amount of plastic already in the ocean would cause problems for centuries. The solution will not come from preventing additional plastic from entering the environment, but instead must come from the safe destruction of plastics already in the environment.

Previous Study on Bacteria and Degradation of Plastics
Many strains of Pseudomonas have been shown to reduce LDPE plastics into PHAs. These are naturally produced by lots of bacteria as a way of storing carbon and energy, however Pseudomonas has a special kind of PHA synthase which can derive PHAs directly from LDPE and PET plastics. Along with physically eating the plastics, bacteria also form a biofilm on them, which lowers their hydrophobic properties and makes them more susceptible to water damage. A study conducted by Bhone Myint Kyaw, Ravi Champakalakshmi, and three others investigated the ability of different strains of Pseudomonas to break down LDPE plastics in a controlled laboratory environment. They stressed that a laboratory environment was not ideal for the degradation of plastics by Pseudomonas, which is known to perform better in real world conditions. The study found that of four strains,

Pseudomonas aeruginosa consumed the most. Over a period of 120 days, it lowered the mass of strips of LDPE plastics by 20%, and the authors of the study predicted that with more time it would continue to reduce the mass at a similar rate. All of the strains reduced the mass by at least 9% in 120 days. This is a promising result, as it shows that Pseudomonas has a lot of potential to break down plastic wastes. However, Pseudomonas is a freshwater bacteria, and is thus unable to survive in the ocean environment.

Proposed Solution
The solution is to create a recombinant organism by incorporating the PHA synthase, and thus the plastic consuming abilities, of Pseudomonas into a bacteria capable of surviving in a saltwater environment. This is possible with the use of plasmids, small rings of DNA that usually code for only a few genes. These are naturally occurring in bacteria, and are expressed and duplicated separately from chromosomal DNA. This allows them to be moved from one organism to another fairly easily, in fact this is a common laboratory experiment for students. Plasmids can be obtained commercially. They contain a gene for resistance to an antibiotic, usually ampicillin, and a place for new genes to be added, called the multiple cloning site.

The chosen recipient of this new DNA is Pseudoalteromonas sp. D41, since it has surface proteins that allow it to bond strongly to ­­ and form biofilm on ­­ hydrophobic materials such as plastics. It is also a common marine bacteria, already present in the environment targeted, and thus will not harm the local ecosystems. Due to its antibacterial properties, it is able to compete well in the formation of biofilms, helping to ensure that it will not be crowded out. All of these traits make Pseudoalteromonas sp. D41 a good candidate.

To help ensure that there is not undesired damage to plastics in use in the ocean, such as buoys and fishing line currently deployed by the aquaculture industry, a control mechanism must be built into the artificial plasmids. They will require the presence of a specific compound, called a ligand, in order to express the gene and produce copies of PHA synthase. Without the presence of the ligand during transcription, mRNA cannot be created from the plasmid, and thus no plastic will be consumed, even if the ligand was previously present. Plastics normally act as a magnet to non­polar organic chemicals. Because of this, the ligand can be incorporated into newly produced plastics which are not designed to survive in the ocean, such as bottles and bags, while being left out of those which should be durable in the ocean. These plastics would slowly release the ligand into the environment, in a similar way to an IUD releasing progesterone over a period of years. When they came into contact with the altered Pseudoalteromonas sp. D41, the ligand would activate the new genes, allowing it to consume the plastic over a period of months. As these plastics eventually made their ways into ocean gyres, they could continue to release the ligand, allowing plastics in the area to also be consumed. Because the period of time it takes for plastic to degrade, even under the expected acceleration, damage to plastics not treated and and in constant exposure to Pseudoalteromonas will be minimal. At the same time, even those plastics that are not treated will experience some degree of accelerated decomposition if they are in the same area as plastics that are treated.

Methods and Results

DNA Integration
DNA does not pass easily through a cell’s membrane, so in order to introduce new plasmids the cells should be kept in a high calcium environment at low temperatures. This creates holes in the membrane, which allow DNA to pass through. Once the cells have holes in them, they can be grown with the new plasmids, then very quickly placed in a 42 degrees C environment, before being chilled again. This is called a heat shock, and it will cause the bacteria to take up the plasmids. To ensure successful integration, the bacteria can then be incubated at 37 degrees C in a sterile solution of water, nutrients, aquarium sea salt, and ampicillin, so that only those who took in the plasmids will survive and multiply.

Preparation for Testing
Once sufficient amounts of bacteria have been obtained, a study can be performed similar to the aforementioned study to test the ability of this new bacteria to degrade LDPE plastics. As with their study, strips of LDPE film from plastic bags will be sterilized by washing in ethanol, then in water, dried, and will be inserted into 30 sterilized falcon tubes filled with a sterile solution of water, nutrients, and aquarium sea salt. Three of these will be set aside as a control. Of the remaining tubes, 18 will be inoculated with altered Pseudoalteromonas sp. D41, and 9 will be inoculated with unaltered Pseudoalteromonas sp. D41, while the control will not be inoculated with anything. Our ligand will be added to 9 of the tubes containing the altered version. All of the tubes will be incubated on a rotary shaker going 120 rpm at 37 degrees C. After 20, 40, and 60 days 3 tubes in each group will be opened to test ­­ along with a control tube, which will be tested for contamination at each of these times. After testing, these tubes and their contents will be discarded. These methods were taken mirroring the test performed on Pseudomonas, and are not expected to mirror the conditions found in nature.

Tests and Results
The first test to be performed is a culture test, to see whether the bacteria is thriving. Swabs will be taken of the inside of the falcon tube, and cultures will be grown in a petri dish with a cellulose pad. Data collected will be number and size of colonies grown over seven days at 37 degrees C. It is expected that some growth will be evident in all but the control tubes. Growth in the control tubes indicates that the samples were contaminated. It is also expected that the number and size of cultures increase with the amount of time the tubes have incubated. The next test will be mass loss of LDPE plastic strips. They will be removed from the solution where they will be first rinsed with water, then washed in SDS, then dried overnight. Their mass will then be measured, and compared to their original mass to determine a percent mass loss. An ideal result would be a loss of mass comparable to that measured with Pseudomonas in those tubes containing both the altered Pseudoalteromonas sp. D41 and the ligand, with minimal loss in the other tubes. The prior study showed an approximately linear increase in mass loss over time, with between 7 and 17 percent mass lost at 40 days.

Any additional observations will be noted, such as apparent change in surface texture, thickness, or color, or the formation of any deposits, such as gas bubbles, or anything else worth noting.

Conclusion

Limitations
The conditions of this experiment are modeled after a previous study, not nature. They are meant only as a proof of concept in the idea that Pseudoalteromonas sp. D41 is capable of obtaining the ability to consume plastics at the same rate as Pseudomonas. It is not as an actual analog to the real environment. If results are successful, additional testing will be required under more realistic conditions. These include lower temperatures, such as would be found in the ocean, as well as lower amounts of available nutrients, and exposure to sunlight and UV rays.

The timeframe under which this experiment can be performed is shorter than that of the previous study, and thus results cannot be compared evenly. Results from the previous study will have to be extrapolated assuming a linear relation for all except the 40 day test.

Implications for the Real World
If results are as hoped, with between 7 and 17 percent of LDPE and PET plastics exposed to Pseudoalteromonas sp. D41 consumed in just 40 days, that would mean that 100 percent of these plastics could be consumed in 8 to 20 months. With the integration of the activating ligand into more and more plastic products, more of these objects would be available to Pseudoalteromonas, and would degrade at this accelerated rate. Even those floating plastics which were not treated would likely experience some degree of acceleration due to their proximity to ligand treated
plastics. Pseudoalteromonas offers a practical solution, since it does not rely on a massive change to the way people live.

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Recombinant Marine Bacteria and the Digestion of Floating Plastic Debris