A recently started project exploring microbial populations attaching to plastic when inmersed in the ocean. Floating plastic (#2, #4, and #5) are deployed in coastal waters for varying times, and DNA extracted for 16S rRNA metagenomic sequencing. Validation of the sampling methodology and preliminary data are shown.

1. Exploring the Microbes attaching to
Plastic Bags in Local Beaches
A community Based Research Project
Ana Maria Barral, Ph.D.
National University
2015

2. Background
• Long-term effect of plastic in the oceans has been
and is studied
• Not much is known what is the effect of plastic
bags on local beaches shorter term
• The “plastisphere” present high content of the
genus Vibrio: pathogens and/or plastic
degraders?
• Hypothesis: floating plastic bags on beaches will
present distinct microbial populations compared
to water.

3. Plastic types tested
Floating plastic types:
• #2 HDPE (produce bags,
milk jugs)
• #4 LDPE (most grocery
bags, juice cartons)
• #5 PP (yogurt & margarine
tubs)
PE: polyethylene
PP: polypropylene

4. Pilot study (March 2015)
• Goal: Validation of the system:
Time frame (4 weeks)
Sterilization methodology
DNA extraction
Sequencing
• Location: Agua Hedionda Lagoon, Carlsbad

5. Pilot location: Agua Hedionda Lagoon
• 400 acre coastal wetland, home to a rich
ecosystem
• Mixture of salt and fresh water

6. Setup of sampling system
• Ocean quality stainless steel cages (316)
• Folded and secured with zip ties
• Attached to a floating device, held approximately
20 inches under surface with weights
Bart’s Iron Design
Capo Beach

7. Sterilization procedure
• Plastic squares approx. 7 cm x 7 cm
• Soaked in 70% EtOH 5 minutes
• UV-irradiated in Germicidal Cabinet
• Stored in sterile autoclave bags
• Cages were autoclaved

8. Experiment Setup
• Day 0 samples: Sterilized plastic controls (directly
frozen from pouches)
• Cages & plastic assembled just before
deployment
• 2 plastic samples per time point (reproducibility)
• Water samples also collected
• Collected weekly for 3 weeks
• Samples placed on ice and stored at -20 oC till
processing

9. Setting up the cages

10. Deploying the samples

11. DNA extraction
• For plastic samples, MO-BIO PowerSoil Kit was
used
• For #2 samples, the whole plastic sample could
be processed
• #4 and #5, smaller pieces
• Yield: 0.09-9 ug/ml (rec. 2 ug/ml)
• For water samples, MO-BIO Power Water

12. 16S rRNA metagenomic sequencing
• Omega Bio-Tek, GA
• Illumina MiSeq platform
• Primers for the V3/V4 region (Klindworth 2013)
• Price tag for 15 samples + 3 controls (free): $1350
• Turnaround time: supposedly 3 weeks, it was over
1 month
• Controls: sterile plastic & no sample (DNA kit),
internal blanks (Omega)

13. DNA reads

14. Samples vs Control

15. Samples vs. Controls

16. Summary system validation
• Good quality DNA was collected to run
metagenomic DNA sequencing
• Blanks and controls had much lower read number
& completely different populations from the
samples => what was collected on plastic came
from the water.
• Replicate read numbers were very similar, except
Cyanobacteria in 2 cases: unequal exposure to
sunlight?

17. Metagenomic data
• Diversity at different taxonomic levels (from
kingdom to species) for each sample
• For ex. plastic #2 at 3rd week:
• Kingdom Phylum

18. Dendogram of genus level classifications
Samples cluster similarly per the sampling day
#2 and #5 are slightly more similar to each other than to #4

19. Main bacterial genera
#2 #4 #5

20. Some interesting results
• Ruegeria lacuscaerulensis is a typical bacterioplankton,
originally described from a lagoon in Iceland. It was
consistently expressed on all three plastic types at all
sampling times.
• Winogradskyella echinorum is a species originally
isolated from a sea urchin.
• Rickettsia marmionii, observed on plastic #2 and #4
during the first week of sampling, has been described to
cause one type of human spotted fever.
• Vibrios were observed at low numbers in all samples

21. Ongoing study (ocean waters)
• More robust design to withstand waves & surf
• Larger cages (lobster trap type) contain the small
sampling cages
• Attached to sturdy buoy or anchored
• Sampling of plastic & H2O for
DNA sequencing
• Swabbing of samples on
culture media

22. Sampling site: Doheny State Beach, Dana Point
In a beach area frequented by
surfers and beachgoers
Close to the San Juan River
mouth
Support from the OC Sheriff’s
Department!

23. Culturing of samples
• To combine genetic
characterization of microbial
populations with morphological
and physiological methods
• 2 media:
– Salt Water Nutrient Agar (general
medium)
– ChromAgar Vibrio specifically for
the isolation & identification of
Vibrio species (95% per CAV)

24. Very preliminary data
• Day 8 samples cultured on Salt Water Agar (24h)

25. More very preliminary data
• Day 8 samples cultured on ChromAgar Vibrio
(24h)
H2O #5
All 3 plastics showed mauve and blue colonies, indicative of V.
parahemolyticus and V. cholerae/V.vulnificus, respectively.

26. What is next?
• Ocean run:
– Results of cultures, possible identification of Vibrio
cultures (?)
– Metagenomic sequencing of plastic and water
– Specific primers for Vibrios or other genera of interest
• Research:
– Increase sampling using a crowdsourced approach
– “Ready-to-deploy” cage sets
– EM of plastic?
– Chemical analysis of plastic?

27. Educational possibilities
• Engaging topic that many care about (ocean,
pollution, water safety)
• Different levels for different courses:
– General Bio lab (experimental design, DNA isolation,
simple culturing)
– Microbiology (cultures, microbial identification)
– Molecular Biology (DNA isolation, PCR)
– Bioinformatics (analysis)

28. Acknowledgments
• Dean Carol Richardson
• Dr. Emelia DeForce
• Dr. Wendy Ochoa
• David Slingluff
• Avi from Bart’s Iron and Jeff from SD Lobsterport
• Michelle Hills and Brett Naftzger
• Sgt. Mike Scalise from OC Sheriff Department
• Dr. James Leichter from SOI
• Dr. Rachel Simmons

29. Questions?