Exploring the Microbes attaching to Plastic Bags in Local Beaches

A community Based Research Project

Ana Maria Barral, Ph.D.National University

2015

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.

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

Pilot study (March 2015)

• Goal: Validation of the system:

Time frame (4 weeks)

Sterilization methodology

DNA extraction

Sequencing

• Location: Agua Hedionda Lagoon, Carlsbad

Pilot location: Agua Hedionda Lagoon

• 400 acre coastal wetland, home to a rich ecosystem

• Mixture of salt and fresh water

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

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

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

Setting up the cages

Deploying the samples

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

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)

DNA reads

Samples vs Control

Samples vs. Controls

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?

Metagenomic data• Diversity at different taxonomic levels (from

kingdom to species) for each sample

• For ex. plastic #2 at 3rd week:

• Kingdom Phylum

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

Main bacterial genera#2 #4 #5

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

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

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!

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 ofVibrio species (95% per CAV)

Very preliminary data

• Day 8 samples cultured on Salt Water Agar (24h)

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.

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?

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)

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

Questions?