This document proposes a research project to study plastic degradation by marine bacteria. The study would collect plastic samples from the ocean, isolate bacteria known to degrade hydrocarbons, and conduct experiments with plastics, seawater, bacteria, and copepods. The experiments aim to determine the bacteria's ability to degrade different types of plastics over time, identify degradation byproducts, and assess copepod survival under different conditions. The results could help understand plastic breakdown in oceans and evaluate whether bacterial activity produces toxic compounds or supports higher life forms.

1. Jacob O’Brien
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Analytical introduction
I chose the Scripps institute as a potential sponsor for this project proposal. In class, when
we had the opportunity to look up sponsors, I found a researcher who would be a perfect match.
Bianca Brahamsha is a researcher with interests including biology and physiology of
cyanobacteria, which are some of the main candidates for plastic degradation. Bacterial diversity
and predator prey interactions are also pivotal in understanding the potential in these research
systems. I am writing a proposal in the format that Scripps suggests. I put why my candidacy
should be considered at the very end of the paper with real life accomplishments that would
probably help in their consideration.
As far as the paper goes, I have really tried to accomplish the ideas we have discussed in
class. The two things that I have really tried to work on this quarter are being specific and paying
close attention to my word choice so that it is as clear and concise as possible. I put forth a lot of
effort into rewriting sentences that were wordy to make them clearer. This has proven difficult,
as I have been writing for years without the knowledge I have obtained from this class. Habits
don’t break so easily in 10 weeks, but I hope that you will see that my writing has improved
since the beginning of the quarter.

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Introduction
According to a report by Plastics Europe, 288 million tons of plastic were produced in
2012 [1]. Much of this ends up in undesirable locations including the oceans. Researchers wish
to find out how much plastic ends up in the oceans because plastics cause damage to the
environment via absorption of toxic compounds and ingestion by marine animals [1]. Ingestion
by higher trophic level organisms is not the ultimate fate for many plastics in the oceans.
Microorganisms exist in complex communities on plastic pieces [2]. Some microorganisms
congregate on the plastics increasing the density and causing them to sink [2]. Others have even
been shown to consume some types of plastic for energy [3, 4, 5]. One day, it may be possible to
take advantage of these microbes to help solve a growing plastic problem in the oceans.
Investigators hypothesize; enrichment of known hydrocarbon degrading microbial communities
by increasing bacterial numbers will increase degradation efficiency. It is also hypothesized that
the byproducts of this degradation will contain toxic compounds in measurable levels.
Background
Estimation of Total Plastics
Eriksen et. al, conducted a large scale experiment to estimate the total plastics present in
the World’s oceans by measuring the number density of different sized plastics. Until their
investigation, the only sampling completed has been on microplastics.
Investigators separated and counted plastics based on their size. “We compared plastic
pollution levels between oceans and across four size classes: 0.33–1.00 mm (small
microplastics), 1.01–4.75 mm (large microplastics), 4.76–200 mm (mesoplastic), and >200 mm
(macroplastic) [1].” Observations were conducted in two ways. First, a net with a mesh size of
0.33 mm was towed behind a boat for between 15 and 60 minutes to collect plastics on and just

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below the surface of the water [1]. This method was utilized 680 times. Then, plastics were
counted, measured, and weighed. Total density was determined by dividing total number by total
area trawled. Second, investigators observed over one side of a boat noting visible debris during
observation times. Observations were conducted 891 times. With the data collected, investigators
entered them into a model to predict a total oceanic distribution of plastics. The model included
important variables to increase the accuracy of the predictions.
The data revealed, conservatively, 5.25 trillion plastic particles weighing 268,940 tons
currently floating in the oceans [1]. Most density estimates were between 1000-100,000 plastic
pieces per square kilometer [1]. Microplastics were the most numerous and the macroplastics
were the heaviest. Most of the plastics were concentrated in the subtropical oceanic gyres [1].
These data are enlightening, but they only scratch the surface. Ultimately, most plastic does not
stay at or near the surface, so what happens to it?
Micro-organismal Association with Plastics
Microorganisms associate with plastics in marine environments. Processes they are
involved in include protection from ultraviolet rays, decreasing buoyancy, attracting higher
trophic level organisms, or even accelerating degradation [3]. One of the main ways plastics
degrade is through photo-degradation. Ultra violet rays break plastics into smaller and smaller
pieces. Some bacteria form a biofilm layer, which promote conducive growing environments.
These biofilms have inherent ultraviolet protection [3]. When enough bacteria congregate on a
piece of plastic, they change the buoyancy of the plastic causing it to sink [3]. Some bacteria
even utilize hydrocarbons as energy sources to break down the plastics [2].

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Carson et al. were interested in finding out what types of bacteria were present on plastics
and in what numbers. They were also interested in finding out how these variables differed based
on variable factors of both the ocean (time, temperature, location) and the plastics (size,
composition). A path was set up from Hawaii to Vancouver, Canada where 17 separate trawl
samples were conducted. A mesh size of 0.33 mm was deployed for one hour at a constant speed
to keep separate measurements equal.
To determine what organisms were present on the samples, plastic pieces were first
rinsed clean of all water and then prepared for scanning electron microscopy. A random degree
of vision was chosen to get an unbiased representation of the samples. Then the microorganisms
were identified based on physical properties. Because of the way samples were preserved, it was
impossible to identify the microorganisms’ species accurately. Identification of the
microorganisms was limited to visual characteristics. Plastic polymer types were also identified.
Every piece of plastic of the 83 samples contained microorganisms. Rod shaped bacteria
and diatoms appeared most frequently on the plastics. Other bacteria types were found
significantly less frequently [3]. The most abundant plastic type was polyethylene. Styrofoam,
which is a form of polystyrene, did harbor significantly more bacillus bacteria compared to the
other plastic types. Surface roughness played a role in harboring diatomaceous life. Samples that
were more rough contained more diatoms. According to the findings, most diversity stems from
the size of the particles compared to the other variables [3]. Now that the general types of
microorganisms that exist on different types of marine plastic debris have been identified, the
next step is to find out more details about these organisms.

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Reisser et al, performed a similar experiment of marine plastic collection. Investigators
collected plastic and identified microbes via scanning electron microscopy. An important detail
observed by researchers was the identification of pits with the shape of these microorganisms in
the plastic debris [6]. These microtextures were indicative of plastic utilization by bacteria.
Larger sizes of plastics as well as these pits allow the possibility for more diverse microbial
colonization. These pits may be important to facilitate further degradation.
Finding Bacteria Capable of Hydrocarbon Degradation
Because the close identification of microorganisms existing on marine plastic pieces was
lacking, Zettler et al, set out to identify these communities. Researchers hypothesized that the
communities existing on plastic are significantly different from those in the surrounding seawater
[2]. Data were collected in a similar fashion to the previous experiment [3], but the samples were
preserved differently to identify the organisms using molecular evidence. Samples were prepared
for scanning electron microscopy as well as Raman spectroscopy, which reveal the composition
of plastics. Investigators extracted DNA from samples, amplified, sequenced, and compared to
known DNA sequences. This process enabled investigators to identify specific bacterial
organisms.
As in the last experiment, most of the plastics identified were polyethylene and
polypropylene which are both common in packaging and single-use applications [2]. The
communities inhabiting the plastics were indeed different compared to the surrounding seawater.
Filamentous cyanobacteria were the second most common morphotype on plastics, but were
completely absent from seawater samples [2]. Diatoms were also present in large numbers on
these plastic pieces. Diatoms are an important find, because they significantly alter plastic
density causing the pieces to sink. An interesting find was the presence of planktonic protists not

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known to associate with any substrates [2]. Investigators observed over a thousand species from
a single fragment of plastic, but most importantly was the identification of some species known
to degrade hydrocarbons. Members of the genera Phormidium, Pseudomonas, and Hyphomonas
were found in significant numbers on plastic debris [2]. All three of these groups have been
shown to be able to degrade hydrocarbons [2]. Members closely related to these three groups
were also identified on debris, which may be another focal point on future studies to determine
the significance of plastic degradation in marine systems.
Hydrocarbon Degrading Capabilities
High-density polyethylene (HDPE) represents up to 64% of synthetic plastics produced
[4]. This type of plastic is an important study point because of its widespread and diverse usage.
Balasubramanian et al, collected HDPE pieces found in the Gulf of Mannar. Investigators
inoculated samples with a synthetic media to facilitate growth of desirable bacteria. After an
incubation period of 12 weeks, bacteria samples were purified and screened for HDPE-degrading
bacteria [4]. To determine the efficiency of bacterial degradation, investigators incubated
samples with a predetermined amount of HDPE. The samples were then washed, dried, and
weighed to determine how much plastic was degraded. Investigators performed a bacterial
adhesion to hydrocarbon assay to determine hydrophobicity levels of these samples. This test
reveals the attraction of bacteria to plastic samples via light spectroscopy. Based on
hydrophobicity, comes the viability of bacterial biofilm. A specific test reveals the presence of a
viable biofilm. Molecular probes attach to cells and fluoresce different colors based on cell
condition. Cells glow red if dead and green if alive. This test allowed investigators to determine
if a biofilm was present on HDPE samples after a 30 day incubation period. Because of the
difficulty involved in accurately measuring population density within biofilms, investigators

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used a more reliable method and estimated bacterial counts by measuring present protein levels.
These proteins were measured and compared to known amounts to determine relative amounts of
bacteria present.
In the experiments, investigators tested 15 different strains of bacteria. Two of the 15
strains were capable of degrading hydrocarbons as the only source of energy [4]. One genus,
Pseudomonas, has been identified in previous studies. The other bacterium, Arhtrobacter, was
not previously identified in reviewed literature. After a 30 day incubation period, there was a
significant decrease in plastic mass. A decrease of 12 percent by Pseudomonas and 15 percent by
Arthrobacter is a promising result. According to the hydrophobicity test, Pseudomonas had a
higher affinity to the plastic, which is interesting because one would think having a higher
affinity would equate to a better degradation coefficient.
Harshvardhan et al conducted a very similar experiment as the one reviewed previously.
One major difference between the two is the type of plastic evaluated. In this experiment, low-
density polyethylene (LDPE) was evaluated. Sixty bacterial samples were evaluated with similar
techniques including identification, hydrophobicity, biofilm viability, and degradation ability. Of
the 60 samples, three of them were identified as capable of degrading the LDPE [5]. The three
useful bacteria identified are Kocuria palustris, Bacillus subtilis, and Bacillus pumilus. The
degradation capabilities of these three bacteria was significantly less than those degrading the
high-density polyethylene with between 1 and 2 percent degradation over a 30 day period. This
is an interesting find, because one would think that with lower density bonds in the plastic,
bacteria would have an easier time utilizing it as an energy source.

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Materials and Methods
The vessel R/V New Horizon, capable of open ocean missions, will be utilized to collect
samples in the Pacific Subtropical Gyre. Samples will be collected via a net with a mesh size of
30μm to capture large and small plastics. Once sufficient samples have been collected, they will
be stored until bacteria will be isolated. Specific members from the genera Phormidium,
Pseudomonas, Hyphomonas, Kocuria, and Bacillus are the target bacteria because of their
potential to degrade hydrocarbons [2, 4, 5]. To isolate bacteria, small samples of seawater with
the plastics will be centrifuged at low g-forces so as to not kill any bacteria, but at high enough
forces to separate them from the plastic. A sterile loop with an approximately 10μl storage
capacity will be dipped into the centrifuged sample to collect suspended bacteria. Next,
investigators will streak plate agar enriched with a mineral medium containing polyethylene and
polypropylene molecules as the only carbon source, because this has been previously shown to
facilitate growth of bacteria capable of degrading hydrocarbons [7]. This method will eliminate
undesired bacteria and ease the identification of desired bacteria. Because the identification is not
guaranteed, the plating technique will be performed several times per sample from several
different samples until desired bacteria are present. Once colonies are isolated, they will be re
streaked to obtain pure isolates.
The first round of experimental samples will be created using one liter of sterile seawater,
sufficient nutrients to support growth, and 50mg each of polypropylene, high-density
polyethylene, and low-density polyethylene. A total of 12 samples will be prepared. One sample
will be a control with only plastics and sterile seawater. One more control sample will contain
plastics, seawater, and an inoculation of bacteria not known to degrade hydrocarbons. The
remaining 10 samples will be inoculated with equal amounts of the four different experimental

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bacteria. All samples will be exposed to the same conditions of 12°C to emulate ambient oceanic
temperatures, a 10 hour day 14 hour night cycle to simulate natural sunlight conditions, and a
constant motion to simulate wave action. After a 30 day testing period, plastics from the samples
will be removed, sterilized, dried and weighed to determine the amount of degradation of each
type. Water from the 10 samples will be mixed thoroughly before exposed to mass spectrometry.
Spectrometry will allow investigators to determine molecular compounds present in the sample
likely to be byproducts of the degradation process. Deciphering the composition of these
compounds will reveal benign versus toxic molecules present.
The second and third round of experiments will be set up almost identical to the first. For
the second round, the same 12 samples will be set up as before only this time adding in a set
amount of copepods to each. The third and final round of experiments will be the same as round
two, except all 12 samples will be inoculated with greatly increased numbers of bacteria. The
samples will undergo the same physical conditions and after a 30-day period, copepod survival
will be evaluated.
Expected Results
After the testing period, a decrease in plastic mass is expected in all experimental groups
compared to control groups. The highest amounts of degradation compared to controls are likely
to take place in the samples inoculated with the most plastic degrading bacteria. This may not be
the case however; too many bacteria may slow the degradation process. Bacteria may be limited
by factors including space, decreasing degradation. It is expected to see a larger decrease in mass
of HDPE compared to LDPE as that is what is shown in previous studies [5]. Because of the
physical properties of polypropylene, which are similar to HDPE, less degradation is expected
compared to either type of polyethylene. However, it is entirely possible polypropylene will have

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higher rates of degradation. It is reasonable to expect this because bacteria are more efficient at
degrading HDPE compared to LDPE.
As well as recording plastic degradation, investigators will also measure copepod
survival in each sample. The lowest survival is expected to be the control samples with only
plastics and seawater, because the lack of sustenance for the copepods. The highest survival will
most likely be the other control samples inoculated with bacteria not associated with plastic
degradation. One possibility of results includes a lower survival of copepods in samples
inoculated with fewer bacteria. This would happen if toxic byproducts were not an important
factor. Less survival would be linked to less nutrition compared to samples with more bacteria.
Another possible set of results would show less survival in the samples inoculated with more
bacteria. If this happened, it may be reasonable to assume the byproducts of plastic degradation
have some effect on the survival of copepods.
Implications
These experiments are important steps to understand the significance of the plastic
problem in the oceans. If results show byproducts of plastic degradation by large amounts of
bacteria are not toxic and can support higher trophic life, then the next step of experiments can
proceed. The next step in the process would be to not only include copepods, but also include
other higher organisms. Copepods may be able to survive, but it is important to know if their
predators can survive in these artificial conditions. Eventually, it will be important to take these
experiments to the field. Real world studies reveal insights not capable of prediction in
laboratory settings because of uncontrollable factors. It is the hope that these results provide the
foundation to a feasible solution to the plastic problem our oceans face.

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Candidacy
I believe I am the most valuable candidate for this research position. I am enthusiastic
about this project because I am fascinated with the microbial world. I have prior experience in
marine microbiology. My time at Saddleback College in Mission Viejo has allowed me to
perform two separate water quality experiments dealing with microbes. I have experience
presenting scientific data both visually and orally. My Bachelor of Science in Biology provides
me with a base knowledge of biological communities. It is my goal to expand on this knowledge
through this project.

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Works Cited
1. Eriksen M. et al, (2014). Plastic Pollution in the World's Oceans: More than 5 Trillion
Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLOS One, 9(12).
2. Henry, C. et al, (2013). The Plastic-Associated Microorganisms of the North Pacific
Gyre. Marine Pollution Bulletin, 75(2), 126-132.
3. Zettler, E. (2013). Life in the "Plastisphere": Microbial Communities on Plastic Marine
Debris. Environmental Science Technology, 47, 7137-7146.
4. Balasubramanian, V. (2014). High-density polyethylene (HDPE)-degrading potential
bacteria from marine ecosystem of Gulf of Mannar, India. Letters in Applied
Microbiology, 51(2), 205-211.
5. Harshvardan, K. et al, (2013). Biodegradation of low-density polyethylene by marine
bacteria from pelagic waters, Arabian Sea, India. Marine Pollution Bulletin,
77(1), 100-106.
6. Reisser, J. (2014). Millimeter-Sized Marine Plastics: A New Pelagic Habitat for
Microorganisms and Invertebrates. PLOSone, 10(1371).
7. Sanchez, O., & Diestra, E. (2005). Molecular Characterization of an Oil-Degrading
Cyanobacterial Consortium. Microbial Ecology, 50(4), 580-588.