ABSTRACT

The project focused on the ability of the coral pathogen Vibrio coralliilyticus to ad-

here to three different kinds of plastic particles, as potential vehicles for the

dispersal on floating plastic debris.

Polyethylene terephthalate (PET), low density polyethylene (LDPE) and

biodegradable polylactide (PLA) were tested as a potential surface material for

Vibrio coralliilyticus biofilm formation by setting up a microcosm with seawater

under two different temperature conditions. It was possible to successfully locate

Vibrio coralliilyticus settled on all three plastic particle surfaces, by means of

scanning electron microscopy and crystal violet staining. The biofilm formation

was documented as confirmed well and by an increase in optical density values

after mechanical detachment of the biofilm and comparing the values to seawater

for reference. The quantitative data from the optical density measurements

suggested that, the floating LDPE particle was preferred for biofilm formation by

Vibrio coralliilyticus.

INTRODUCTION

Out of 80 million tons of plastics produced as consumer packing material,

approximately 10–20 million tons per year end up in the oceans, where they

persist and accumulate [1],[2]. Like all surfaces in the marine environment micro-

plastics, are rapidly colonized by microorganisms creating a specific environment,

that differs from the surrounding water communities [3]. Sequencing results

obtained from recent studies indicate that, potentially pathogenic Vibrio might be

present on floating plastic debris [3]. Furthermore, these particles might function

as vectors for the distribution of pathogens across the ocean [3]. One of these

potential pathogens might be Vibrio coralliilyticus, which causes tissue damage in

several coral species, known as the coral bleaching [4].

RESULTS

The biofilm formation of Vibrio coralliilyticus was documented by measuring the

optical density at 600 nm. Plastic particles from the microcosm, were placed into

seawater and after a mechanical detachment of the biofilm, the turbidity was

measured. For reference seawater from the microcosm was measured without

biofilm detachment. Referring to the graph in figure 1, biofilm detachment from

the LDPE particle shows mostly the highest optical density values. The values for

both temperatures have similar results, however after detaching the biofilm the

values for all three kinds of particles were higher compared to seawater.

A photographic documentation is illustrated in figure 2, showing the plastic

particles after they were dyed in a crystal violet solution. The particles were

previously incubated for one week in filtered or unfiltered seawater with Vibrio

coralliilyticus .There are obvious differences visible with the violet coloring

between the samples from the negative control, only containing filtered seawater

and the samples with V.coralliilyticus inoculation. The results suggest that the

V.coralliilyticus strain is able to form biofilms on the PET, LDPE and PLA

particles.

To locate the V.coralliilyticus on the plastic particle and confirm the data from the

crystal violet staining a scanning electron microscopy was performed. The

electron-microscopic pictures shown in figure 3, lead to the same conclusion that

V.coralliilyticus settles on all three kinds of plastic particles.

Figure 3: The photo documentation of the scanning electron microscopy (S3400, HITACHI).

The pictures show Vibrio coralliilyticus settled on the different kinds of plastic particles:

polylactide (top), polyethylene terephthalate (middle) and low density polyethylene (bottom).

Electron acceleration voltage = 10,0 kV for all pictures taken. The magnification varies

between 35×103 and 27×103 times.

Further experiments in this project:

Vibrio coralliilyticus show a growth rate of maximal 0,28 generations h-1, in the

alkaline seawater (pH 8) under 30°C incubation. Additionally, by means of

confocal laser scanning microscopy it was possible to locate genetically modified

Vibrio cells that have previously integrated a plasmid with a green fluorescent

protein (GFP) gen by the triparental mating technique.

CONCLUSION

The usage of small scale portions of seawater in laboratory glass tubes as a

microcosm might not be able to mimic living conditions in the ocean, but the

results strongly suggest that Vibrio coralliilyticus is able to adhere on plastic

surfaces introduced to seawater. The pictures from the scanning electron

microscopy as well as the crystal violet staining show V.coralliilyticus cells on all

three kinds of analyzed plastics (PET, LDPE, PLA). Therefore, plastic particles

theoretically provide the right surface structure for the settlement and a long

distance distribution through hitch-hiking is conceivable. Although, one must keep

in mind that V.coralliilyticus has to compete with other bacteria in natural

conditions, and might not be able to prevail against the other communities on the

plastic surfaces, what has to be further examined.

The optical density measurements in the first and the second approach showed a

possible preference of V.coralliilyticus towards the LDPE particle, that is the only

floating particle analyzed in this project and thereby provides the most air contact.

The underlying cause for this observation is probably that facultative anaerobic

Vibrio species are able to produce more adenosine triphosphate by switching to

the aerobic respiration in presence of oxygen.

REFERENCES [1] Andrady, A.L.,(2003). Plastics and the environment.Publisher: John Wiley and Sons, ISBN 0-471-09520-6

[2] Gourmelon, G.,(2015). Global Plastic Production Rises, Recycling Lags. Worldwatch Institute

[3] Kirstein, I. V, Kirmizi, S., Wichels, A., Garin-fernandez, A., Erler, R., & Martin, L.,(2016). Dangerous hitchhikers ? Evidence for potentially

pathogenic Vibrio spp . on microplastic particles. Marine Environmental Research, 120, 1–8.

[4] Ben-haim, Y., Zicherman-keren, M., & Rosenberg, E. (2003). Temperature-Regulated Bleaching and Lysis of the Coral Pocillopora damicornis

by the Novel Pathogen Vibrio coralliilyticus. Applied and Environmental Microbiology, 69(7), 4236–4242.

Plastic Pellets as potential Shuttles for the Transport of

Vibrio coralliilyticusTheresa Felix

Bachelor-Thesis, Molecular Bioanalytics

Principal: René Prétôt, University of Applied Science Northwestern Switzerland

Expert: Dr. Simon Hebeisen, CSO at B’SYS GmbH

Supervisor: Dr. Moritz Müller, Swinburne University of Technologies Sarawak

Figure 1: Bar chart with the mean from the

optical density values (at 600 nm) after

detaching the biofilm, by “vortexing”, in

comparison to seawater. The

measurements were performed three

times, on subsequent days with biological

triplicates in double determination. PET=

polyethylene terephthalate; LDPE = low density polyethylene and PLA= polylactide

Figure 2: Crystal violet staining of the plastic

particles. The pictures at the top: plastic

particles from the negative control without

bacteria inoculation in filtered seawater. The

pictures in the middle: plastic particles under

the same condition, but with V.coralliilyticus

inoculation in filtered seawater. The pictures

at the bottom: the plastic particles from

unfiltered seawater with V.coralliilyticus

inoculation. Pictures were taken by a

stereomicroscope with 5x magnification.

Picture series in A) low density polyethylene

(LDPE); B) polylactide (PLA ) and in C)

polyethylene terephthalate (PET).

pla

pet

ldpe

Po

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e te

rep

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w d

ensi

ty p

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2µm 1µm 2µm

2µm2µm2µm

2µm1µm2µm

A B C

0,0364

0,0485 0,04830,0454

0,0000

0,0100

0,0200

0,0300

0,0400

0,0500

0,0600

Water PET LDPE PLA

Op

tica

l den

sity

/600

nm

Sample type

First measurement (25 C)

0,0414 0,04400,0467 0,0452

0,0000

0,0100

0,0200

0,0300

0,0400

0,0500

0,0600

Water PET LDPE PLA

Op

tica

l den

sity

/600

nm

Sample type

First measurement (30 C)

0,0415 0,0428 0,0430 0,0427

0,0000

0,0100

0,0200

0,0300

0,0400

0,0500

Water PET LDPE PLA

Op

tica

l den

sity

/600

nm

Sample type

Second measurement (30 C)

0,0376 0,0382 0,0383 0,0382

0,0000

0,0100

0,0200

0,0300

0,0400

0,0500

Water PET LDPE PLA

Op

tica

l den

sity

/600

nm

Sample type

Second measurement (25 C)

0,0387 0,0404 0,0403 0,0406

0,0000

0,0100

0,0200

0,0300

0,0400

0,0500

Water PET LDPE PLA

Op

tica

l den

sity

/600

nm

Sample type

Third measurement (25 C)

0,0364 0,0387 0,0389 0,0385

0,0000

0,0100

0,0200

0,0300

0,0400

0,0500

Water PET LDPE PLAOp

tica

l den

sity

/ 6

00

nm

Sample type

Third measurement (30 C)

A B

C D

E F